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Contains fulltext : 251844.pdf (Publisher’s version ) (Open Access) OBJECTIVES: Neoadjuvant chemotherapy and radical cystectomy (RC) are underutilized standards of care for the treatment of muscle-invasive bladder cancer (MIBC) due to high patient burden from systemic toxicities and postoperative complications, respectively. TAR-200 is a novel intravesical drug delivery system developed to release gemcitabine into the bladder urine continuously, resulting in distribution of drug into stromal layers of the bladder. The primary aim of the TAR-200-101 study was to evaluate the safety of TAR-200 in patients with MIBC prior to RC (NCT02722538). METHODS AND MATERIALS: This phase I, open-label study was conducted across 6 US and European sites. Eligible patients were aged ≥18 years with histologically confirmed T2a-T3b N0-N1 M0 urothelial cancer and had refusal or were ineligible to receive cisplatin-based combination chemotherapy. Two arms were enrolled serially. Patients in Arm 1 had residual tumor >3 cm after transurethral resection of bladder tumor (TURBT); those in Arm 2 had undergone maximal TURBT (residual tumor

A game bird producer in the North Central region of the United States submitted unhatched ring-neck pheasant (Phasianus colchicus) eggs for diagnostic evaluation. The submitting complaint was a drastic drop in hatchability. This operation has its own breeder birds that are housed in outside pens. This hatch occurred in the latter third of the production cycle. Typical hatchability for this operation is around 75% (± 3%). The hatchability of this hatch was between 14%-15%. Approximately 30,000 eggs were set with an expected hatchability of about 23,000 birds. The number of birds from this hatch was less than 4500, with a net loss approaching 20,000 chicks. All unhatched eggs submitted were in late stage development. The chick embryos had pipped through the shell but died before hatching. Approximately 5000 eggs originating from an outside breeder source were also set at the same time in the same machines and experienced a normal hatch. The exterior surfaces of the eggshells of the unhatched eggs experiencing low hatchability were swabbed and submitted for bacteriologic evaluation. Additionally, embryos from some of the unhatched eggs were removed aseptically from their eggshells, and their internal organs were harvested and submitted for bacteriologic evaluation. The bacteriology results identified no pathogenic bacteria from the eggshells. However, the embryo samples revealed large quantities of Enterococcus faecalis. In discussions with the producer, the only factor identified was an unusually warm period followed by an atypically cold and wet period during the time of egg collection for those eggs experiencing low hatchability.

The biosecurity of composting as an emergency disposal method for cattle mortalities caused by disease was evaluated by conducting full-scale field trials begun during three different seasons and using three different envelope materials. Process biosecurity was significantly affected by the envelope material used to construct the composting matrix. Internal temperatures met USEPA Class A time/temperature criteria for pathogen reduction in 89%, 67%, and 22%, respectively of seasonal test units constructed with corn silage, straw/manure, or ground cornstalks. In trials begun in the winter, survival times of vaccine strains of avian encephalomyelitis and Newcastle disease virus were noticeably shorter in silage test units than in the other two materials, but during summer/spring trials survival times in ground cornstalk and straw/manure test units were similar to those in test units constructed with silage.

Traditional gemcitabine bladder instillations (40 mg/mL) exhibit limited efficacy possibly due to micturition and saturable nucleotide uptake limiting tumor exposure. The present study investigates gemcitabine's anti-tumor and immunomodulatory effects after continuous low dose intravesical administration to enhance bladder tumor exposure. The direct effects of low dose gemcitabine at nominal bladder urine concentrations of 0, 20, 40, or 80 µg/ml were studied in athymic nude rats with human MIBC cells, T24-TurboFP635, injected into the bladder. After 3 days tumor growth, gemcitabine or vehicle was perfused into the bladder over 5 days, N=6 per group. Tumor bioluminescence was measured on day 5. A companion study in immunocompetent Wistar rats inoculated with syngeneic NBTII bladder tumors provided tumors for immunocyte analysis. A significant reduction in bladder tumor bioluminescence of 47.6%, 70.5% and 91.2% vs control was observed in the 20, 40, and 80 µg/ml groups, respectively. Flow cytometric analysis of residual tumors in immunocompetent rats demonstrated a significant decrease of 85.2% in the proportion of tumor T regulatory cells to T effector cells. Gemcitabine immunogenicity was studied in immunocompetent rats with NBTII cells injected into the bladder and subcutaneous flank tissue; flank tumors were implanted either simultaneously with bladder tumors or 14 days after bladder tumor implantation. Control groups received only flank tumors, N=15, or bladder and flank tumors with vehicle perfusion, N=10. Treatment groups, N=10, received two 5-day bladder perfusions, yielding gemcitabine urine concentrations of 10, 20, and 40 µg/ml which were separated by 7 days. Flank tumor growth was similar with tumor doubling times of 4.5 days after subcutaneous injections of 1.0, 2.5, or 5.0 x 106 cells and was unchanged with concomitant bladder tumor implantation and vehicle perfusion. During the first gemcitabine perfusion cycle, flank tumor growth was fully arrested, with tumor sizes averaging only 50% of controls in rats with concomitant bladder tumors; this effect was maintained during the 7-day drug free period in the 60 µg/ml group. Initiation of the second perfusion resulted in rapid flank tumor ablation in all but one animal completing the study. A similar priming effect was observed in the 15 and 30 ug/ml groups. Delaying flank tumor implantation to 3 days before the second perfusion not only prevented tumor growth but completely ablated all tumors. The results suggest continuous low dose intravesical gemcitabine inhibits bladder tumor growth, shifts the tumor microenvironment toward effector T cell dominance and induces a systemic immune response sufficient to suppress distant disease. Perhaps for the first time, the results also demonstrate a cytotoxic agent can induce a significant abscopal response when administered under proper conditions. Citation Format: Dennis H. Giesing, Don Reynolds, Vikas Agarwal, Chris Cutie, Balaji Ramachandran, Aravindakshan Jayaprakash, Satheesh Rajendiran, Purnand Sarma. Significant cytotoxic and immumomodulatory effects of continuous low dose intravesical gemcitabine in rodent bladder tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-122.

Campylobacter jejuni is a gram-negative enteric organism causing gastroenteritis in humans (38). As a major food-borne pathogen, C. jejuni is well adapted in its mammalian and avian hosts, as well as in food animal production environments. So that it can survive in different conditions, C. jejuni has evolved multiple strategies for adaptation, including high rates of genetic variation (mediated by mutation and horizontal gene transfer) and differential gene expression (6, 11, 30). Indeed, previous analyses of the genomic sequences of C. jejuni revealed the presence of multiple genes encoding regulatory functions (9, 14, 32, 35). The majority of the transcriptional regulators have not been functionally characterized, but the two-component regulatory (TCR) systems in C. jejuni have recently received attention. In C. jejuni NCTC 11168 and RM1221, each system has nine response regulators and six histidine sensor kinases (9, 32). Several of the regulators or TCR systems, including DccRS (28), PhosSR (49), FlgSR (50), CbrR (36), RacRS (4), and CheY (53), have been studied, and all were found to be required for Campylobacter colonization in vivo. The RacRS system is responsive to temperature and controls the expression of multiple proteins in C. jejuni, while the PhosSR system senses phosphate conditions and modulates the expression of 12 genes that are involved in phosphate transport and utilization (4, 49). DccRS controls the expression of several genes encoding probable membrane-associated proteins and is required for Campylobacter colonization of mice and chickens, but the signals to which it responds and the functions of the DccR-regulated genes have not been defined (28). CbrR modulates the Campylobacter response to bile, but its cognate sensor kinase and the target genes controlled by it are unknown (36). The FlgSR system controls the flagellar regulon and affects the motility of Campylobacter (50). It was also found that FlaR undergoes phase variation due to the presence of homopolymeric tracts of adenine and thymine in the coding gene (13). These examples illustrate that C. jejuni utilizes multiple TCR systems for adaptation to different environments. In addition to the TCR systems, several non-TCR system regulators, including Fur, SpoT, HspR, and CmeR, have also been characterized in Campylobacter. Fur functions as a transcriptional repressor and controls iron homeostasis in C. jejuni (31, 45). Mutation of Fur affected the expression of 53 genes and significantly reduced the colonization of chickens by Campylobacter (31). HspR is a negative regulator for the heat shock response system in C. jejuni, and inactivation of HspR led to increased expression of several genes involved in the heat shock response and decreased expression of 17 genes (1). The HspR mutant showed decreased motility, increased sensitivity to temperature, and reduced adherence to and invasion of cultured epithelial cells (1). SpoT functions as a regulator for the stringent response in C. jejuni and is important for the survival of Campylobacter under various stress conditions (11). Deletion of spoT resulted in differential expression of multiple genes and reduced Campylobacter adherence, invasion, and intracellular survival in cell cultures (11). In a previous study by workers in our laboratory (21), a transcriptional regulator designated CmeR was characterized. CmeR belongs to the TetR family of transcriptional regulators and functions as a repressor of CmeABC, a resistance-nodulation-division-type efflux pump (23). The CmeABC pump is composed of three membrane components (CmeA, CmeB, and CmeC) and contributes to Campylobacter resistance to various antimicrobial agents and bile compounds present in the intestinal environment (23, 24). Inactivation of CmeABC abolished the ability of C. jejuni to colonize chickens (24), indicating that bile resistance is an important physiological function of CmeABC and that this efflux pump plays an important role in facilitating Campylobacter adaptation to the intestinal tract. CmeR is encoded by a gene located immediately upstream of the cmeABC operon and has two distinct domains, an N-terminal helix-turn-helix (HTH) DNA-binding motif and a potential ligand-binding domain in the C-terminal region (21). An in vitro electrophoretic mobility shift assay showed that CmeR binds specifically to the inverted repeat (TGTAAT) in the promoter region of cmeABC and represses the transcription of this efflux operon. Deletion of CmeR or mutation in the CmeR-binding site impedes the repression and results in overexpression of CmeABC (21). Importantly, the expression of cmeABC is strongly induced by bile salts in culture media (22). Since bile compounds are normally present in animal intestinal tracts, it is likely that CmeABC is upregulated during in vivo infection. Indeed, the DNA microarray work conducted by Stintzi et al. showed that cmeABC was significantly upregulated in rabbit ileal loops (40). Real-time quantitative reverse transcription-PCR (qRT-PCR) conducted in our laboratory also showed that there was a 14-fold increase in the expression of cmeABC in the chicken cecum compared with an in vitro culture (Y. W. Barton and Q. Zhang, unpublished data). Bile salts inhibit the binding of CmeR to the promoter DNA of cmeABC and thus release the inhibition of cmeABC by CmeR (22), resulting in overexpression of cmeABC. Recent protein crystallization studies confirmed the two-domain structure of CmeR and showed that CmeR functions as a homodimer (12). A striking feature of CmeR revealed by crystallization is the presence of a large ligand-binding pocket, which has the capacity and flexibility to accommodate diverse ligands, including bile salts. This finding suggests that CmeR may interact with multiple ligands in modulating gene expression in C. jejuni. Despite the recent advances in our understanding of the structure and function of CmeR and its transcriptional regulation of cmeABC, it is still unclear if CmeR regulates the expression of other genes in C. jejuni. It is also not known if CmeR is important for Campylobacter colonization in vivo. In this study, we compared the transcriptomes of C. jejuni NCTC 11168 and an isogenic CmeR mutant of this strain using a DNA microarray along with other molecular methods and identified multiple genes regulated by CmeR. We also characterized a gene (Cj0561c) that is highly repressed by CmeR and determined the role of CmeR in Campylobacter colonization in the chicken model system. Our new findings indicate that CmeR is a pleiotropic regulator modulating the expression of multiple genes with diverse functions in Campylobacter and is required for optimal colonization of chickens.

The dose proportionality of deflazacort was assessed following single-dose oral administration at doses of 3, 6, and 36 mg to 24 healthy young adult volunteers. The active metabolite of deflazacort (21-desacetyl deflazacort) was monitored in plasma using a sensitive, semi-microbore liquid chromatographic method. Cmax averaged 10.4 +/- 5.0, 19.8 +/- 7.5, and 132.6 +/- 52.5 ng mL-1 for the 3, 6, and 36 mg doses, respectively. AUC(0-infinity) averaged 38.5 +/- 37.1, 64.9 +/- 20.8, and 411.7 +/- 148.5 ng h mL-1 for the same three doses, respectively. Elimination half-life ranged from 1.9 +/- 0.5 h at the 6 mg dose to 2.4 +/- 1.5 h at the 36 mg dose. Regression analyses of dose versus Cmax and AUC(0-infinity) yielded intercepts which were not significantly different from zero (p > 0.05) and slopes which were significant (p 0.05). These data indicate that deflazacort exhibits dose-proportional pharmacokinetics.

Two 21-d experiments were conducted to document the effects of an early immunologic stress and changes in dietary ME(n) on selected characteristics of immune function of newly hatched turkeys. Eight treatments were included in each experiment. Treatments were the result of complete factorial arrangements of two types of injection and four isonitrogenous diets. Turkeys in both experiments were injected i.p. with .5, .5, and .2 mL of saline (SAL) or .5, .5, and .2 mL of a solution of Escherichia coli lipopolysaccharide (LPS) (100 micrograms LPS/mL SAL) at 1, 3, and 5 d of age, respectively. In Experiment 1, two diets were formulated to contain 2,800 kcal ME(n)/kg. One was a corn-soybean meal based diet (CSBM) and the other contained 8% Solkafloc (SKF). A third diet (3,100 kcal ME(n)/kg) was formulated by substituting 8% sucrose (SUC) for the 8% SKF. The fourth diet (HE) included in Experiment 1 was formulated to contain 3,700 kcal ME(n)/kg. The CSBM and SUC diets and two additional diets were tested in Experiment 2. The latter were the CSBM diet containing 74.5 mg ibuprofen/kg (IBU) and a corn-soybean meal diet formulated to contain 3,100 kcal ME(n)/kg (CS31). Concentrations of plasma IgG and jejunal IgG and IgA were not affected by injection or diet. Age-related changes in Ig concentrations were consistently observed in Experiments 1 and 2. Injection with LPS reduced the number or responses of blood leukocytes to mitogens at 8 d of age (P < .01), as compared with samples from turkeys injected with SAL. Leukocytes in whole blood samples from turkeys fed the HE diet responded less to LPS stimulation than those fed the SUC diet (P < .01). Injection with LPS did not markedly affect the characteristics of immune function studied, and feeding a diet with 3,100 kcal ME(n)/kg and 28.5% crude protein did not measurably affect the characteristics of immune function of young turkeys.

Eight day-old male and female ringneck pheasants (Phasianus colchicus) were inoculated with group D rotavirus and necropsied at 4, 7, and 11 days post-inoculation. The intestinal tracts were examined by light and electron microscopic and immunohistochemical methods. By 4 days post-inoculation, 213 (66%) inoculated birds were stunted and had diarrhea and dilated intestines. Intestinal villi were shortened, and many villous enterocytes were partially detached from the lamina propria. Crypts were hyperplastic, and the lamina propria contained a diffuse infiltrate of lymphocytes, plasma cells, and macrophages. Immunoreactivity to rotaviral antigen was localized to enterocytes on the tips of villi in the duodenum, jejunum, and proximal ileum. By 7 days post-inoculation, 3/3 (1 00%) inoculated birds had clinical signs and gross and microscopic changes similar to those at 4 days post-inoculation but more severe. Immunoreactivity was localized in enterocytes scattered along the sides of villi, in occasional crypt enterocytes, and within macrophages in the villous lamina propria. Ultrastructurally, infected enterocytes contained cytoplasmic aggregates of viroplasm with multiple viral core particles. Numerous mature virions (60-75 nm in diameter) were present within dilated components of the cytocavitary network. Macrophages within the lamina propria contained phagocytosed remnants of necrotic virus-infected cells. By 11 days post-inoculation, birds did not have gross lesions, but 1/2 (50%) had mild crypt hyperplasia and an infiltrate of leukocytes in the lamina propria. Occasional enterocytes along the sides of villi and macrophages in the lamina propria were immunoreactive for viral antigen. Group D rotavirus is an enteropathogen in pheasants and causes intestinal lesions similar to those caused by enteric rotaviral infections in other species. Kqv words; Enteritis; immunohistochemistry; intestine; pheasants: rotavirus.

The separation of the four stereoisomers present in Beraprost sodium, a prostacyclin analogue, has been accomplished using an α1-acid glycoprotein stationary phase (Chiral AGP column). The stereoisomers are baseline resolved with a runtime of less than ten minutes. This chiral separation is used to quantitate the four Beraprost sodium stereoisomers present in the bulk drug, tabletted formulations and in pharmacological and toxicological studies. The mobile phase variables that were found to have an effect on the stereoisomeric separation were studied and include: ionic strength, type and concentration of organic modifier, mobile phase pH and column temperature. Optimum stereoisomer separation was achieved on the Chiral AGP column. Calibration curves were linear for all four stereoisomers over the range of 0.024 to 4.04 μg/ml using fluorescence detection (correlation coefficients were greater than 0.999). A detection limit of 0.004 μg/ml was found for each stereoisomer. This assay has been used to determine the ratio of the four stereoisomers in the bulk drug as well as in the final formulated tablets.

The irradiation of 2-ethoxy-1,4-naphthoquinone with allyl alcohol and 3-butenol affords diols containing the novel spiro[3.3]heptane unit.

A sensitive and selective liquid chromatographic method to quantitate MDL 201,012 in human plasma was developed and validated. MDL 201,012 (I), diethyl-MDL 201,012 (internal standard, II) and desmethyldiol-MDL 201,012 (masking agent, III) were isolated from basified plasma (2 mL) by solid phase extraction using Bond-Elut C-18 cartridges. Endogenous components were selectively removed prior to eluting the analytes from the sorbent. Components were separated using on-line LC column switching with a cyanopropyl precolumn and a phenyl analytical column. The analytical column effluent was monitored electrochemically at a glassy carbon electrode at a potential of +1025 mV vs. Ag/AgCl. Peak-height ratios were proportional to the amount of MDL 201,012 added to plasma over the range 125-7500 pg/mL MDL 201,012. Absolute recovery of MDL 201,012 from human plasma was > 94% across the calibration range. The minimum quantitation limit was 125 pg/mL. Assay precision (%RSD) ranged from 5.2 to 13% based on the analysis of quality control standards containing 125, 250, 500, 1000, 2500, 5000 and 7500 pg/mL MDL 201,012. Corresponding assay accuracy (% relative error) was +/- 8.5%. The method has been successfully used to quantitate MDL 201,012 in samples from acute dose tolerance studies in human volunteers.

CmeABC, a multidrug efflux system in Campylobacter jejuni, plays an important role in the resistance to different antimicrobials and toxic compounds. Although this efflux system has been well characterized in C. jejuni and to a less extent in C. coli, it is unknown if CmeABC homologs are functional in other Campylobacter spp. In this study, the cmeABC homologs were identified and functionally characterized in five Campylobacter species including C. jejuni, C. coli, C. lari, C. upsaliensis, and C. fetus. Our results indicated that cmeABC is present in all five Campylobacter spp. and the genomic organization of this efflux operon is similar among the Campylobacter spp. Insertional mutagenesis of cmeB increased the susceptibilities of all the five Campylobacter spp. to structurally diverse antimicrobials. Together, these results indicated that the CmeABC efflux system is conserved at both the genomic and functional levels in all five Campylobacter spp. examined in this study, further highlighting the significant role of CmeABC in Campylobacter pathobiology.

The irradiation of 2-ethoxy-1,4-naphthoquinone with allyl alcohol and 3-butenol affords diols containing the novel spiro[3.3]heptane unit.

A 3-year study was conducted in Iowa to evaluate the feasibility of using composting for emergency disposal of cattle mortalities. During the study, 49 metric tons of 450 kg cattle carcasses were composted in 27 replicated unturned windrow test units constructed during three different seasons of the year. Each test unit contained 1.8 metric tons of carcasses enveloped in one of 5 different materials: corn silage, ground cornstalks, straw/manure, leaves, or a soil/compost blend. Due to their water absorbing capacity and ability to evaporate absorbed water, the volume of leachate released into the soil was generally less than 5% of the 500-600 mm of precipitation that fell on the test units. Chemical analysis of 1.2 m deep soil cores collected from beneath the composting test units prior to and following composting showed statistically significant increases in chloride concentrations at all depths beneath composting test units constructed from silage, cornstalks, straw, and the soil/compost blend. Statistically significant increases in % total carbon (silage test units only) and % total nitrogen (silage, cornstalk, straw/manure test units) were limited to the top 15 cm of soil. Increases in these pollutants were moderate, amounting to less than 5X, 0.2X and 0.4 X respectively of chloride, % total carbon, % total N concentrations prior to composting. Statistically significant increases in total ammonia-nitrogen were noted at depths of up to 90 cm beneath test units constructed with silage or leaves, and at 30 cm and 15 cm depths respectively beneath test units constructed with straw/manure and cornstalks. The ammonia-nitrogen increases were large, ranging from 40-160 X of pre-composting levels of ammonia in the topsoil. When compared with the groundwater pollution potential of carcass burial, however, the estimated total mass of N contained in the composted cattle carcasses was 4-10 X the increases in total N measured in the soil beneath the composting test units.

Moisture affects the physical and biological properties of compost and other solid state fermentation matrices. Aerobic microbial systems experience different respiration rates (oxygen uptake and CO2 evolution) as a function of moisture content and material type. In this study the microbial respiration rates of 13 compost-bulking materials were measured by a pressure sensor method at 6 different moisture levels. The experimentally determined respiration quotient (RQ) values were used to calculate CO2 respiration rates from O2 consumption. The RQ values of all materials were around 1.0 except for silage, oat straw and leaves which were about 1.5. A wide range of respiration and heat production rates were observed for different materials, with alfalfa hay, silage, oat straw, and turkey litter having the highest values. These four compost-bulking agents may be particularly suitable for improving internal temperature and pathogen destruction rates for disease-related mortality composting. Optimum moisture content was determined based on measurements across a range that spans the maximum respiration rate. A mechanistic model of moisture kinetics was also used to predict the optimum moisture levels. There was good agreement between experimental observations and modeled optimum moisture content. The optimum moisture content of each material was observed near WHC, which ranged from near 65 to over 85% on a wet basis for all materials except a highly stabilized yard waste compost (optimum around 30% w.b.). This study demonstrates the importance of moisture content on the biodegradability of organic materials and specific respiration rates of each material. The results can be used to develop moisture management and process control strategies to maintain compost and cover materials in an acceptable range.

Physical and biological parameters of corn stalks, silage, and oat straw were determined and the variation of airflow characteristics and biological activity were evaluated under varying

Bacteriophage therapy represents a potential alternative to the use of antibiotics to control proliferation of pathogenic bacteria. As an alternative to the strategy where a limited number of doses of large numbers of lytic bacteriophages are administered, a novel method delivery system was developed so that phages are continually released into the culture. Specifically, a non-pathogenic Escherichia coli strain was constructed that was lysogenic for a lytic mutant of bacteriophage lambda. This lysogen was shown to be effective at decreasing the number of lambda-sensitive E. coli in vitro. Construction of this E. coli strain was accomplished by development of a plasmid-based system utilizing the site-specific recombination machinery of bacteriophage P22 to integrate DNA constructs into the host chromosome. This recombination system is useful for strain construction and other genetic manipulations in both E. coli and Salmonella enterica serovars.

Carcass degradation rate, environmental impacts, and bio-security of windrow-type composting test units were monitored in replicated seasonal trials to assess the feasibility of using composting for emergency disposal of cattle and other large livestock carcasses. Internal temperatures were highest in test units constructed with corn silage. Test units constructed with ground cornstalks or straw and manure were generally 10-20 °C cooler. O2 concentrations in the core of ground cornstalk test units typically exceeded 15%, while those in corn silage and straw/manure test units were in the 5-10% range during the initial weeks of the trials. Despite differences in core temperature and O2 concentration, soft tissue degradation rates were the same in all test units, taking 4-6 months in units constructed during warm weather, and 8-10 months during cold-weather. It is believed that the less favorable (lower) temperatures in the cornstalks may have been offset by significantly higher O2 concentrations which favor rapid aerobic decomposition. Thirty to 45 cm of cover material proved effective in absorbing and retaining odorous gases and leachate. Odors samples collected from the surface of the mortality composting piles typically had low threshold values (< 1500) that differed little from odors emitted by stockpiles of the cover material alone. Leachate volumes were

A sensitive and selective liquid chromatographic procedure to quantitate the deflazacort metabolite 21-hydroxy-deflazacort (DF-21OH) in human plasma was developed and validated. DF-21OH and fludrocortisone acetate (internal standard, IS) were isolated from human plasma (2 mL) by solid-phase extraction onto C-18 cartridges. Potential interferences were selectively removed and analytes were eluted with ethyl acetate. Following evaporation, the residue was reconstituted for HPLC analysis. Separation was achieved by gradient elution using a 5 microns YMC Basic column (2.0 x 100 mm) with mobile phases consisting of 20% methanol and 50% acetonitrile in 50 mM phosphate buffer (pH 3) at a temperature of 50 degrees C. Flow rate was maintained at 0.3 mL/min., and analytes were quantified spectrophotometrically at 246 nm. The assay was validated over the range 1.0 to 500 ng/mL DF-21OH. Calibration curves were prepared using a weighted (1/concentration) nonlinear quadratic regression algorithm. Peak-height ratios were proportional to the amount of DF-21OH added to plasma. Assay precision (%RSD) ranged from 4.2 to 11%, with a corresponding assay accuracy (% relative error) of +/- 2.8%. Absolute recovery of DF-21OH from plasma was 78-86% over the concentration range. The minimum quantitation limit was 1.0 ng/mL.

Two experiments were conducted to determine the effect of removing bacteria, including long segmented filamentous organisms (LSFO), from inoculum known to induce stunting syndrome (SS) in poults. Experiment 1 consisted of two identically designed trials. In each trial, each of four treatments was assigned to an isolator. Three treatments consisted of dosing, by crop intubation, groups of 1-day-old poults with unfiltered SS inoculum or filtrate of inoculum passed through .45- or .20-micron microfilters. Uninoculated poults were dosed with inoculum carrier, saline. Experiment 2 was done in battery facilities. Three rooms were used and each room housed one of three treatment groups. Triplicate pens of 10 poults each within each room were dosed by crop intubation with saline (uninoculated), unfiltered inoculum, or filtrate from .20-micron filtration. As compared with uninoculated poults, weight gain through 7 days was reduced 20% (P less than .05) by unfiltered and filtered inocula in both experiments. Jejunal maltase activity also was decreased (P less than .01) by unfiltered and filtered inocula. Feed efficiency (FE) was not determined in Experiment 1, but in Experiment 2, FE from 1 to 14 days of age was impaired by inoculum, irrespective of filtration. This effect was not evident during the 14- to 20-day period. The observation that the adverse effects of giving filtered inoculum to poults were the same as those caused by unfiltered inoculum indicated that bacteria, including LSFO, were not primary causative agents of the SS experimentally induced in poults.

PD 117519 (I) [(R)-iV-(2,3-dihydro-lH-inden1-yl)adenosine, CI-9471 is an adenosine agonist which has shown oral antihypertensive activity in pharmacological animal models [l, 21. Physicochemical properties of this compound include an aqueous solubility of 0.21 mg ml-‘, a p& value of 2.4 (in 50% methanol), and an apparent octanol-water partition coefficient (log P) of 1.99 at pH 7. A sensitive and selective assay was required to quantitate PD 117519 in dog plasma to support pre-clinical pharmacokinetic studies.

Two experiments were conducted to determine the influence of Streptococcus faecium M-74 supplementation of broiler diets. Mixed-sex or male only chicks were used in the experiments. Both experiments consisted of six dietary treatments and four replicates per treatment in a randomized block design. The S. faecium was fed alone for 21, 36, or 44 days or for 44 days with S. faecium supplemented in the water for the first 14 days. In another treatment, S. faecium was fed for 44 days, but the feed was restricted for Days 8 through 13. The S. faecium was also fed in combination with antibacterial products (AP) for 44 days. An additional diet was an unsupplemented basal, and another was supplemented only with AP. In Experiment 1, feed efficiency was significantly better with the basal and diets supplemented with S. faecium than with those diets supplemented with AP or AP and S. faecium. In Experiment 2, BW of broilers at 44 days of age were significantly heavier for broilers receiving 5. faecium in the feed and also S. faecium in the water for the first 14 days as compared with broilers receiving AP or AP and S. faecium supplementation. There were no significant differences in carcass yield or composition characteristics. In Experiment 1, the scores representing S. faecium colonies found in the intestinal tract were not influenced by dietary treatment. The ceca had the highest S. faecium score of any of the intestinal tract locations. In Experiment 2, broilers receiving diets containing 5. faecium or the basal had significantly more S. faecium colonies in the ceca than the broilers receiving the diets containing AP or AP and S. faecium. The S. faecium colony counts in the duodenum, jejunum, and ileum were significantly lower on Days 14 and 21 than on Day 7. The S. faecium counts from the ceca were significantly lower on Day 21 only. The numbers of S. faecium colonies in the duodenum, jejunum, ileum, and ceca were increased on Day 29. The ceca also consistently yielded the highest S. faecium counts.

Conventional nested and TaqMan reverse transcription-polymerase chain reaction (RT-PCR) assays for the detection of avian pneumovirus (APV) were evaluated and compared with virus isolation (VI) for sensitivity and specificity. Respiratory tissues and tracheal swabs were collected from experimentally inoculated turkeys between 1 and 21 days postinoculation (DPI) and tested by all detection methods. APV was detected by both RT-PCR procedures as early as 1 DPI and as late as 17 DPI, whereas virus was isolated only between 3 and 7 DPI. Pooled tracheal swab supernatant and dry swabs were excellent specimens for the detection of APV between 3 and 8 DPI. Turbinate and sinus specimens were the most productive samples over the entire collection period. Both RT-PCR assays were rapid and more sensitive than VI for the detection of APV in tissue and swab specimens from infected turkeys. RT-PCR allows for the rapid detection of APV from a variety of respiratory tissues as well as from dry swabs and tracheal swab supernatants. Antibody to APV was detected in 50% of the sampled APV-inoculated birds at 8 and 9 DPI by enzyme-linked immunosorbent assay (ELISA). Early seroconversion (8-10 DPI) allows antibody detection to be used as a screening tool for APV. Rapid and sensitive detection methods are needed for APV, a highly contagious disease affecting U.S. poultry.

SUMMARY. A reverse transcription-polymerase chain reaction (RT-PCR) assay for the detection of avian pneumovirus (APV), Colorado strain (US/CO), was evaluated for sensitivity and specificity. The single-tube RT-PCR assay utilized primers developed from the matrix (M) gene sequence of the US/CO APV. The RT-PCR amplified the US/CO APV but did not amplify other pneumoviruses, including the avian pneumoviruses subgroups A and B. The RT-PCR was capable of detecting between 100.25 mean tissue culture infective dose (TCID50) and 10-0-44 TCID50 of the US/CO APV. These results have demonstrated that the single-tube RT-PCR assay is a specific and sensitive assay for the detection of US/CO APV.

The fluorescent antibody (FA) assay was developed for detecting the stunting syndrome agent (SSA) from intestinal tissue. Similarly, the indirect fluorescent antibody (IFA) assay was developed for detecting serum antibodies to SSA. Convalescent antiserum from turkeys orally immunized with SSA was found to be the primary antibody of choice for the FA assay. Intestinal jejunal samples from poults inoculated 3-4 days postinoculation (DPI) was found to be the best antigen source for the IFA assay. SSA was detected from the intestinal tracts of experimentally inoculated birds at 2 DPI with highest level of reactivity at 3 DPI by the FA assay. After 4 DPI the level of SSA infectivity of the intestines waned to low levels. Serum antibody was detected from experimentally inoculated birds as early as 7 DPI with all birds tested seroconverting by 12 DPI. These assays should prove useful for future studies concerning stunting syndrome.

Stunting syndrome is an enteric disease of young turkeys that results in reduced growth (stunting) of poults and impaired feed efficiency. A virus, which has been termed the stunting syndrome agent (SSA), causes stunting syndrome. In this study passive immunity was evaluated as a method of protecting poults from stunting syndrome. One-day-old poults were injected with either tryptose phosphate broth, an anti-SSA antibody preparation, or an anti-Newcastle disease virus antibody preparation before challenge by placing them into SSA-contaminated isolators or control (nonchallenge) isolators. Poults that received anti-SSA antibodies were significantly heavier (P0.05) and did not display as severe clinical disease compared to birds that did not receive the anti-SSA antibodies. However, the birds that received anti-SSA antibodies and were challenged were significantly lighter (P0.05) than birds that were not challenged. The results of this trial demonstrate that the injection of anti-SSA antibodies benefited poults undergoing stunting syndrome. The role of passive immunity, either through breeder hen vaccination or through supplying antibodies to poults artificially (i.e., at the hatchery), may have future applications in alleviating stunting syndrome.

Studies were performed to determine if passive immunization with hyperimmune sera generated to specific Newcastle disease virus (NDV) proteins conferred protection against virus challenge. Six groups of 3-wk-old chickens were passively immunized with antiserum against either hemagglutinin-neuraminidase/fusion, (HN/F) protein, nucleoprotein/phosphoprotein (NP/P), Matrix (M) protein, a mixture of all NDV proteins (ALL), intact ultraviolet-inactivated NDV (UVNDV), or negative sera. Blood samples were collected 2 days postimmunization, and the birds were challenged with Texas GB strain of NDV. Antibody titers were detected from those recipient birds that had received the antisera against the HN/F, ALL, or UVNDV by a hemagglutination inhibition test, an enzyme-linked immunosorbent assay (ELISA), and a virus neutralization test. Antibodies were detected only by the ELISA from the birds that had received antisera against NP/P and M protein. Antibody titers in the recipient birds dropped by two dilutions (log2) after 2 days postinjection. Birds passively immunized with antisera against HN/F, ALL, and UVNDV were protected from challenge, whereas chickens passively immunized with antisera against NP/P and M protein and specific-pathogen-free sera developed clinical signs of Newcastle disease. The challenge virus was recovered from the tracheas of all passively immunized groups. The presence of neutralizing antibodies to NDV provided protection from clinical disease but was unable to prevent virus shedding from the trachea.

SUMMARY. The role of cell-mediated immunity (CMI) in protection of birds from Newcastle disease was investigated by two different strategies in which only Newcastle disease virus (NDV)-specific CMI was conveyed without neutralizing antibodies. In the first strategy, selected 3-wk-old specific-pathogen-free (SPF) birds were vaccinated with either live NDV (LNDV), ultraviolet-inactivated NDV (UVNDV), sodium dodecyl sulfate-treated NDV (SDSNDV), or phosphate-buffered saline (PBS) (negative control) by the subcutaneous route. Birds were booster vaccinated 2 wk later and challenged with the velogenic Texas GB strain of NDV 1 wk after booster. All vaccinated birds had specific CMI responses to NDV as measured by a blastogenesis microassay. NDV neutralizing (VN) and hemagglutination inhibition (HI) antibody responses were detected in birds vaccinated with LNDV and UVNDV. However, birds vaccinated with SDSNDV developed antibodies that were detected by western blot analysis but not by the VN or HI test. Protection from challenge was observed only in those birds that had VN or HI antibody response. That is, birds with demonstrable CMI and VN or HI antibody response were protected, whereas birds with demonstrable CMI but no VN or HI antibody response were not protected. In the second strategy, birds from SPF embryos were treated in ovo with cyclophosphamide (CY) to deplete immune cells. The birds were monitored and, at 2 wk of age, were selected for the presence of T-cell activity and the absence of B-cell activity. Birds that had a significant T-cell response, but not a B-cell response, were vaccinated with either LNDV, UVNDV, or PBS at 3 wk of age along with the corresponding CY-untreated control birds. The birds were booster vaccinated at 5 wk of age and were challenged with Texas GB strain of NDV at 6 wk of age. All birds vaccinated with LNDV or UVNDV had a specific CMI response to NDV, VN or HI NDV antibodies were detected in all CY-nontreated vaccinated birds and some of the CY-treated vaccinated birds that were found to have regenerated their B-cell function at 1 wk postbooster. The challenge results clearly revealed that CY-treated birds that had NDV-specific CMI and VN or HI antibody responses to LNDV or UVNDV were protected, as were the CY-nontreated vaccinated birds. However, birds that had NDV-specific CMI response but did not have VN or HI antibodies were not protected from challenge. The results from both strategies indicate that specific CMI to NDV by itself is not protective against virulent NDV challenge. The presence of VN or HI antibodies is necessary in providing protection from Newcastle disease.

SUMMARY. The effect of cyclophosphamide (CY) treatment in ovo on avian B and T cells was studied. CY was injected in ovo on the 16th, 17th, and 18th days of incubation. Blood samples were collected periodically from CY-treated and nontreated birds after hatch and were used to measure blood lymphocyte responses to the T-cell and B-cell mitogens, concanavalin A and lipopolysaccharide (LPS), respectively. Additionally, flow cytometric analysis was used to determine the presence of B and T cells in peripheral blood, and birds were vaccinated with Newcastle disease virus (NDV) antigen at 3 wk of age and booster vaccinated at 5 wk of age. CY treatment reduced hatchability by 35%-40%, increased mortality by 3%-5% within the first 2 wk of life, and induced a significant retardation in body weight gains. At 2 wk of age, approximately 50% of CY-treated birds were devoid of B-cell mitogenic responsiveness while demonstrating significant T-cell mitogenic responsiveness. However, Bcell responses were observed at 4 and 6 wk from a small percentage of birds that were originally T-cell responsive and B-cell nonresponsive at 2 wk of age. Flow cytometric analysis of peripheral blood lymphocytes revealed that CY-treated birds had significantly less B cells (or were devoid of B cells) than the corresponding nontreated control birds. However, no significant difference in the T-cell percentage was observed between CY-treated and nontreated birds. CY-treated birds did not produce detectable antibodies specific for NDV during the first and second weeks postvaccination, as demonstrated by hemagglutination inhibition assay. However, antibodies were detected in some CY-treated birds 10 days postbooster. Those antibody-positive birds were found to be the same birds that had subsequently responded to the LPS mitogen on the blastogenesis microassay. This study indicates the importance of monitoring the B- and T-cell responses in CY-treated birds to identify those birds in which B-cell regeneration may have occurred.

Five continuous cell lines, swine testicular (ST), human rectal tumor (HRT 18), fetal rhesus monkey kidney (MA104), bovine turbinate (BT), and quail tracheal (QT35), were evaluated and compared with chicken embryo fibroblasts (CEFs) for their ability to propagate B1 or Texas GB strains of Newcastle disease virus (NDV). The NDV Texas GB strain replicated in all the continuous cell lines used in this study. Only the ST and QT35 cells produced a cytopathic effect (CPE) similar to that produced in CEFs. However, the ST cell line remained attached while displaying CPE, whereas infected QT35 cells detached, as did the CEFs. The B1 strain of NDV replicated in ST cells, MA104 cells, and CEFs but with less CPE as compared with the Texas GB strain. Pretreatment with trypsin did not enhance CPE with either NDV strain at the level tested. Sera evaluated for neutralizing antibody titers to NDV were significantly higher in titer when the ST cell line was used and compared with CEFs. A high correlation was found between the microscopic examination and the tetrazolium dye (MTT) microassay methods for determining the viral neutralization endpoint, thus suggesting the ST cell line and MTT microassay could be used as an alternative to CEFs and microscopic examination for evaluating neutralizing antibodies titers to NDV.

SUMMARY. Mitogenic responsiveness of intestinal intraepithelial lymphocytes (i-IEL) to concanavalin A (Con A), phytohemagglutinin P (PHA-P), and lipopolysaccharide (LPS) from Salmonella typhimurium were evaluated in various age groups of turkeys by a colorimetric blastogenic microassay. Comparisons were made between mitogenic responses of turkey iIEL and peripheral blood lymphocytes (PBL). The results from this study demonstrated that i-IEL and PBL of turkeys responded to T-cell mitogens, Con A and PHA-P, in every age group examined. The LPS induced a significant mitogenic response in PBL but not in i-IEL of turkeys. The mitogenic responses of turkey i-IEL and PBL to the three mitogens examined were similar to mitogenic responses observed in an earlier study performed by using chicken i-IEL and PBL. The results indicated a difference in mitogenic response between different age groups. An increase was found in mitogenic response of i-IEL to both T-cell mitogens from 3 days of age to 1 wk of age, whereas mitogenic response of PBL to all three mitogens declined significantly from 1 day of age to 3 days of age. The highest mitogenic response of i-IEL to T-cell mitogens was observed at 1 wk of age. The highest mitogenic response of PBL to both T-cell mitogens was observed at 1 day of age and the highest PBL response to LPS was observed at 16 wk of age. The mitogenic response induced by PHA-P provided less variability between age groups than the mitogenic response induced by Con A.

A reverse transcription-polymerase chain reaction assay was developed for the detection of avian pneumovirus (Colorado strain) (APV-Col). The specific primers were designed from the published sequence of the matrix protein gene of APV-Col. The primers amplified a product of 631 nucleotides from APV-Col. The assay identified only APV-Col and did not react with Newcastle disease virus and infectious bronchitis virus.

A colorimetric assay employing MTT (3-[4,5-dimethylthiazole-2-yl], 2-5-diphenyltetrazolium bromide) was used to determine the mitogenic response of intestinal intraepithelial lymphocytes (i-IELs) of chickens to T- and B-cell mitogens. Comparisons between mitogenic responses of i-IELs and peripheral blood lymphocytes (PBLs) were made to examine potential relationships. The results from this study indicated that T-cell mitogens, concanavalin A (Con A), and phytohemagglutinin-P (PHA-P) induced mitogenic stimulation in i-IELs. Although stimulation indexes of both i-IELs and PBLs were similar, the optical densities (ODs) of i-IEL cultures containing Con A or PHA-P were 20- to 50-fold lower than the ODs of PBL cultures containing the same mitogen. The lower conversion of MTT to formazan resulting in lower ODs in i-IEL cultures indicated a lower level of cellular activity in the i-IELs than in the PBLs. The mitogenic responses of both i-IELs and PBLs to Con A and PHA-P were dose dependent. The responsive concentration of Con A for i-IELs was within the range of 25-50 micrograms/ml, whereas the responsive concentration of PHA-P for i-IELs was 50 micrograms/ml. Three days of incubation was found to be adequate to induce a significant (P < 0.05) mitogenic response for both T-cell mitogens. Lipopolysaccharide was unable to induce a mitogenic response in i-IELs, which was attributed to the lack of B cells in the i-IEL population. This technique may prove useful in evaluating and studying the role of i-IELs in local cell-mediated immune responses of the gastrointestinal tract.

Stunting syndrome (SS) is a viral enteric disease of turkey poults. The etiologic agent (stunting syndrome agent [SSA]) of this disease has been reported recently. The objective of this study was to develop a method for in vitro propagation of SSA. Primary cells, various continuous cell lines, and embryonated eggs were evaluated. Turkey embryos that were inoculated via the amniotic cavity at 24-25 days of incubation were susceptible to SSA infection. The jejunal maltase activity of SSA-inoculated turkey embryos was significantly (P < or = 0.001) lower than that of control embryos. D-xylose absorption was also altered in SSA-infected turkey embryos. The extent of reduction of D-xylose absorption and maltase activity in the infected embryos was nearly identical to that observed when day-old poults were infected with SSA. The intestines from the infected turkey embryos were pale, thin walled, and distended with fluid. Electron microscopic examination of the intestinal fluid and epithelial cell lysate of infected embryos revealed pleomorphic membraned SSA viral particles. SSA that had been serially passaged in turkey embryos retained its ability to induce SS in day-old poults. All the primary and continuous cells that were evaluated did not support the replication of SSA on the basis of cytopathic effects, electron microscopy, and turkey embryo inoculation. Inoculation of chicken embryos by various routes failed to support SSA. All routes of inoculation, other than the amniotic route at 24-25 days, failed to support SSA in turkey embryos. The results of the this study indicate that the SSA was successfully propagated in turkey embryos that exhibited alterations in embryo intestinal absorption and digestive enzyme activity similar to poults with SS. Successful propagation of SSA in turkey embryos should prove beneficial for future studies including characterization of SSA, prevention and control strategies, and enteric disease modeling.

high morbidity including reduced growth, impaired feed efficiency, and diarrhea. The etiologic agent of this disease has not been previously reported. The objectives of the present study were to identify, isolate, and purify the etiologic agent of SS. Day-old poults were orally inoculated with a SS-inducing inoculum. The intestinal epithelial cells (IECs) were isolated on the fourth day postinoculation. The IECs were lysed and filtered through 0.2, 0.1-, and 0.02-[xm filters. The cell lysate filtrate (0.1 $zm) was subjected to density gradient ultracentrifugation. Intact IECs, filtrates from IECs (0.2, 0.1, and 0.02 Izm), and IEC lysate fractions from gradients (FRG) were used as inocula to infect day-old turkey poults. The weight gain, jejunal maltase activity, and gross intestinal lesions were used as the parameters of evaluation. Weight gain and maltase activity were reduced (P - 0.001) by the isolated IECs, 0.2 and 0.1 ,um filtrates, and FRG when compared with corresponding controls. IEC lysate filtrate (0.1 Rm) and FRG were examined under transmission electron microscope (EM). Enveloped, pleomorphic particles varying in size from 60 to 95 nm were observed and termed stunting syndrome agent (SSA). Primary cultures of turkey IECs were used to further isolate and propagate the SSA. Following the fifth passage in the turkey IECs, the cell lysate induced SS in day-old poults. SSA particles were observed under EM after the fifth passage. The results of this study provide evidence that a viral agent has been isolated and identified from IECs of SS-infected poults and is the etiologic agent of SS.

SUMMARY. Primary cell culture has been widely used in various types of studies and proven useful for the isolation and identification of avian pathogens. Difficulties in growing intestinal epithelial cells in vitro have limited their use for such studies. In the present study, a co-culture system was developed for the primary culture of intestinal epithelial cells. A monolayer obtained from 14-to- 16-day-old turkey embryo intestinal fibroblasts was used as a feeder layer. Feeder layers from turkey embryo fibroblasts and from a continuous cell line (mouse 3T3 fibroblasts) were also employed but were not as successful. The intestinal epithelial cells were isolated by dissociation from the intestinal tracts of 1-day-old turkey poults and grown on the feeder layers. Growth and maintenance media were supplemented with various components, incuding fetal calf serum, chicken serum, hormones, and other growth factors. The epithelial cells grown on feeder layers from the intestinal fibroblasts allowed the intestinal epithelial cells to be maintained in vitro for periods of 7 to 10 days. This technique may prove useful for various applications, incuding isolation of enteropathogens, and for basic studies of the intestinal tract concerning such subjects as physiology, immunology, and toxicology. RESUMEN. Cultivo celular primario de celulas epiteliales intestinales de pavo. Los cultivos de celulas primarias han sido ampliamente usados en varios tipos de estudios y son muy utiles para el aislamiento e identificaci6n de pat6genos aviares. Las dificultades en el crecimiento de celulas epiteliales intestinales in vitro han limitado su uso en tales estudios. En el presente estudio se desarrollo un sistema de cocultivo para celulas epiteliales intestinales primarias. Una monocapa obtenida de fibroblastos intestinales de embriones de pavo de 14 a 16 dias de edad fue usada como monocapa alimentadora. Las monocapas alimentadoras obtenidas de fibroblastos de embriones de pavo y de lineas celulares continuas (fibroblastos de rat6n 3T3) fueron tambien empleadas pero sin ningun exito. Las celulas epiteliales intestinales fueron aisladas del tracto intestinal de pavitos de un dia de edad por disociaci6n y cultivadas sobre las monocapas alimentadoras. El medio de crecimiento y de mantenimiento fue suplementado con varios componentes incluyendo suero fetal bovino, suero de pollo, hormonas y otros factores de crecimiento. Las celulas epiteliales cultivadas sobre las monocapas alimentadoras de fibroblastos intestinales permitieron a las celulas intestinales epiteliales ser mantenidas in vitro por periodos de 7 a 10 dias. Esta tecnica puede ser util para varias aplicaciones incuyendo el aislamiento de enteropat6genos y para estudios basicos del tracto intestinal relacionados con areas tales como fisiologia, inmunologia y toxicologia.

SUMMARY. The head-associated lymphoid tissues of the chicken, composed of the harderian gland and the conjunctiva-associated lymphoid tissue (CALT), were studied to determine whether changes occurred in lymphocyte subpopulations as chickens age from 1 week to 8 weeks. The B cells and subpopulations of T-lymphocytes of the head-associated lymphoid tissues were identified using in situ immunohistochemical staining. Monoclonal antibodies specific for various lymphocyte surface antigens were used. The concentration of T-lymphocytes, particularly CD3+ and CD4+ cells, within the harderian gland increased with age, whereas the concentration of B cells remained the same. B-lymphocytes were observed within the germinal centers of the CALT of 4-week-old birds. The T-lymphocytes within the CALT surrounded the B-cell-rich germinal centers. CD3+ T-lymphocytes were the predominant cell population in all age groups examined. Increasing concentrations of CD3+, CD4+, and CD8+ T-lymphocytes were observed within the CALT as chicks developed from 1 week to 4 weeks of age. Finally, no changes were observed in lymphocyte populations within the CALT as chicks developed from 4 weeks to 8 weeks of age.