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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.

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.

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.

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.

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 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.

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 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.

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. 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.

In three separate experiments, 2- or 5-day-old commercial turkey poults were inoculated orally with astrovirus and examined for clinical signs and for gross and microscopic lesions over a period of 14 days. By day 2 postinoculation (PI), inoculated poults had developed diarrhea, generalized loss of intestinal tone, and dilated ceca that contained light-yellow fluid feces and gas; these changes persisted through day 10 PI. Mild crypt hyperplasia was the only change discernible by light microscopy, and it was first noted in the proximal jejunum on day 1 PI, in the distal jejunum and ileum on day 3 PI, and in the duodenum on day 5 PI. A significant (P < 0.05) increase in crypt depth and area was documented by image analysis on day 3 PI. Ultrastructural evaluation revealed intracytoplasmic aggregates of astrovirus in enterocytes on the sides and base of villi in the ileum and distal jejunum on day 3 PI. Based on the findings, it was concluded that astrovirus caused lesions and replicated in both upper and lower segments of the small intestine in turkey poults.

SUMMARY. A blastogenesis microassay employing 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide (MTT) was adapted to measure blastogenic responses of lymphocytes from the chicken's head-associated lymphoid tissues (i.e., the harderian gland and conjunctiva-associated lymphoid tissue) to T- and B-cell mitogens. Lymphocytes isolated from peripheral blood, spleen, and the harderian gland had highly significant (P < 0.01) responses to T- and B-cell mitogens compared with control lymphocytes cultured without mitogens. Cultured lymphocytes obtained from the harderian gland had highly significant mitogenic responses to the T-cell mitogen concanavalin A (25 to 100 ,g/ml) and to the B-cell mitogen Salmonella typbimurium lipopolysaccharide (1.25 to 5.0 Mg/ml) compared with the control lymphocytes. Mitogenic responses of cultured lymphocytes obtained from the conjunctivaassociated lymphoid tissue could not be measured within the given parameters of the blastogenesis microassay. This was primarily due to the low yield of lymphocytes, which proved to be a limiting factor. The ability of the MTT blastogenesis microassay to detect blastogenic responses of the harderian gland to mitogens may be indicative of its usefulness for measuring cell-mediated immunity responses to other antigens. RESUMEN. Respuestas mitogenicas del tejido linfoide asociado a la cabeza en los pollos. Se adapto una micro prueba blastogenica usando 3-(4,5-dimetiltiazol-2-yl)-2,5 bromuro de tetrasodio difenilico (MTT), para medir las respuestas blastogenicas de los linfocitos del tejido linfoide asociado a la cabeza de los pollos (glindula de Harder y tejido linfoide asociado a la conjuntiva) frente a los mit6genos de las celulas B y T. Los linfocitos aislados de la sangre periferica, bazo y la glandula de Harder tuvieron respuestas altamente significantes (P < 0.01) a los mit6genos de las celulas B y T, comparadas con los linfocitos controles cultivados sin mitogenos. Los linfocitos cultivados obtenidos de la glandula de Harder, comparado con los linfocitos controles, tuvieron respuestas mitogenicas altamente significantes a la concalabalina A, mitogeno de las celulas T a dosis de 25 a 100 jg/ml, lo mismo que al lipopolisacirido de Salmonella typhimurium, mitogeno de las celulas B, a dosis de 1.25 a 5.0 jig/ ml. Las respuestas mitogenicas obtenidas de los linfocitos cultivados del tejido linfoide asociado a la conjunctiva no pudieron medirse dentro de los parfimetros dados en la micro

pheasant chicks was determined to be a group D rotavirus based on its RNA electropherotype and its serologic relatedness to known turkey group A and D rotaviruses using immune electron microscopy and agar-gel immunodiffusion assays. Further characterization revealed a buoyant density in cesium chloride of 1.347 g/cm3 for double-shelled particles and 1.365 g/cm3 for single-shelled particles. The average size for the pheasant-origin group D rotavirus was determined to be 80 nm for double-shelled particles and 70 nm for single-shelled particles. In contrast, the average size for a turkey group A rotavirus isolate was determined to be 73 nm for double-shelled particles and 60 nm for single-shelled particles. There was no evidence of viral replication by the pheasant-origin group D rotavirus (either cytopathic effect or increase in numbers of viral particles) after six passages in MA104 cells. The hemagglutinating activity of the pheasant origin group D rotavirus was compared with that of a turkey origin group A rotavirus. Differences and similarities were noted depending upon the species of erythrocytes employed and the temperature of incubation. RESUMEN. Caracterizaci6n de Rotavirus del grupo D. Se determin6 que un rotavirus aislado del contenido intestinal de faisanes de cuello anillado

SUMMARY. One-day-old turkeys were inoculated per os with material shown previously to induce stunting syndrome (SS). Weight gain and feed efficiency of inoculated poults from 1 to 13 days of age were impaired (P < 0.01) compared with uninoculated poults. Examination of the jejunal mucosa by scanning and transmission electron microscopy showed the presence of long-segmented filamentous organisms (LSFOs) in poults that had been inoculated with SS. These organisms were not seen in jejuna of uninoculated poults. Further research is needed to characterize LSFOs and to determine their involvement, if any, in the adverse effects associated with SS.

Over the last decade, numerous publications have appeared describing \ analyses for ascorbic acid in food products, pharmaceuticals, and biological samples. This review focuses on the chemistry associated with many of these procedures. The papers discussed have historical importance, are important to understanding the method, or have significantly advanced ascorbic acid analysis. The review has 4 major, sections: spectroscopic, electrochemical, enzymatic, and chromatographic methods of analysis.

An automated liquid chromatographic assay using unattended robotic sample preparation has been developed and validated to quantitate 3,7-dimethoxy-4-phenyl-N-1H-tetrazol-5-yl-4H-furo(3,2-b)indole-2- carboxamide (CI-922) in human plasma. The drug and internal standard 2 were isolated from plasma by solid-phase extraction onto Bond-Elut C-18 cartridges. Plasma interferences were selectively removed, and the compounds were eluted from the cartridge. Separation was achieved at ambient temperature using a C-18 column (5 microns, 4.0 X 150 mm) with an isocratic eluant consisting of acetonitrile:water:glacial acetic acid (45:55:1). The column effluent was monitored spectrophotometrically at 340 nm. Peak-height ratios were proportional to plasma concentrations of 1 over the range 0.025-5.0 micrograms/mL, and the recovery of 1 from plasma was 63.5%. Calibration curve precision was +/- 6.7%, based on relative standard deviations (RSD) of 1.9 to 6.7% for calibration standards. The accuracy of calibration curves was +/- 7.2%, with relative errors ranging from -7.2 to 3.6%. Assay precision was +/- 4.8%, based on RSD values of 3.9, 4.8, and 3.4% for spiked control samples of pooled human plasma containing 0.20, 1.50, and 2.50 micrograms/mL of 1, respectively. Assay accuracy was -0.7 to 2.0%. This method has been used to analyze 1 in clinical plasma samples.

Intestinal content samples from four flocks of pheasant chicks experiencing diarrhea and increased mortality were evaluated using immune electron microscopy and genome electropherotyping techniques. Enteric viruses serologically and electropherotypically indistinguishable from turkey rotaviruses and turkey rotavirus-like viruses (RVLVs) were detected. Rotavirus-only infections, RVLV-only infections, and simultaneous rotavirus and RVLV infections were demonstrated in these flocks.

Four flocks from one commercial market-turkey operation in Ohio were monitored for the presence of enteric viruses. Each flock was sampled at intervals from placement until at least 7 weeks of age; sampling was more frequent in the first 4 weeks of life. The earliest infections detected were astrovirus infections or combination infections of astrovirus and rotavirus-like virus (RVLV) or astrovirus and rotavirus. During the first 4 weeks of life, astrovirus was the most frequently detected virus, followed by RVLV, then rotavirus. These viruses were seldom detected beyond 4 weeks of age. In three of the four flocks, no viruses were detected in samples collected before 6 days of age; in one flock, however, rotavirus and astrovirus were identified from samples collected at 3 days of age. Experimental infection of specific-pathogen-free poults with astrovirus and RVLV produced enteric diseases in poults and demonstrated that astrovirus was shed into the intestinal tract before RVLV. Poults experimentally infected with astrovirus and RVLV displayed clinical signs of diarrhea and upon necropsy exhibited dilated ceca, frothy gaseous intestinal contents, and loss of intestinal tone.

Virus particles of 30 nm diameter and star-shaped morphology were detected in intestinal contents of turkey poults and were identified as astroviruses. Seventy-six intestinal samples from 65 commercial turkey flocks between 6 and 35 days of age were evaluated for the presence of astroviruses by immune electron microscopy. Astroviruses were frequently detected in intestinal samples from poults that had enteritis and diarrhea of undetermined etiology. Astroviruses were geographically widespread and were present in poults from all six operations evaluated. Astroviruses were inoculated into specific-pathogen-free poults. Changes observed in the gastrointestinal tract were: dilatated ceca containing yellowish frothy contents, gaseous fluid in the intestinal tract, and loss of tone of the intestinal tract (gut thinness). Poults experimentally inoculated with astrovirus gained significantly less body weight and absorbed significantly less D-xylose than uninoculated controls.

Turkey rotaviruses from the intestinal contents of poults were isolated and serially propagated in MA104 cell monolayers by a simple procedure. The initial virus isolation was done by low-speed centrifugation of the inoculum onto the monolayers, and subsequent passages were accomplished in roller-tube monolayers using trypsin-treated virus suspensions. Each of the turkey rotavirus isolates possessed the morphologic, antigenic, and genomic attributes characteristic of turkey group A rotaviruses. Attempts to isolate and serially propagate turkey rotavirus-like viruses in MA104 cell monolayers by this procedure were unsuccessful. Turkey reoviruses also did not serially propagate in MA104 cell monolayers by this procedure.