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Broiler chicks were spray treated on the day of hatch with titrated dosages (10(6), 10(7), or 10(8) anaerobic CFU) of a characterized competitive exclusion culture (CF3) and challenged orally on day 3 with 10(4) CFU of Salmonella typhimurium. On day 10, cecal contents from control and CF3-treated chicks were cultured for S. typhimurium to determine the minimal efficacious dosage of the CF3 culture. The experiment was repeated in three replicated trials. Resistance to Salmonella cecal colonization was dosage related and progressively enhanced at the 10(7)- and 10(8)-CFU dosages compared with the 10(6)-CFU dosage. The 10(7)-CFU dosage was selected as the minimal effective dosage and evaluated for efficacy during a 43-day broiler growout study. Six hundred broilers were spray treated on the day of hatch and compared with 600 controls. One-half of the control and CF3-treated birds were challenged orally on day 3 with 10(4) CFU of S. typhimurium and designated "seeders." The remaining unchallenged birds were designated "contacts." Compared with the controls, the recovery of Salmonella cells from the ceca of the CF3-treated broilers was significantly decreased (P0.01) in the challenged seeders on days 21 and 43 of growout. Salmonella contamination of floor pen litter was significantly reduced (P0.05) in pens of CF3-treated birds compared with controls. The transmission of Salmonella cells from seeder to contact birds in the same pens was decreased significantly (P0.01). The results indicated that treatment of broiler chicks on the day of hatch with the 10(7)-CFU dosage of CF3 culture effectively increased resistance to S. typhimurium challenge during growout to market age.

Experiments were conducted to evaluate the effects of different competitive exclusion (CE) cultures on the concentration of cecal propionic acid in 3-day-old broiler chicks, and the correlation between cecal propionic acid concentration and protection againstSalmonellacolonization. CE cultures that significantly (P 0.05) increase cecal propionic acid concentrations in 3-day-old chicks failed to protect (P > 0.05) against cecalSalmonellacolonization in 10-day-old chicks compared with untreated controls. A significant (P

An 81-d-old continuous-flow (CF) culture of broiler cecal bacteria was maintained in a lactose-based broth. The culture had been previously proven effective against Salmonella colonization in young chicks, especially when the chicks were provided dietary lactose. Portions of the CF culture were batch-cultured in glucose- and lactose-based broths containing 14C-labeled lactose, glucose, galactose, or lactic acid to determine the effect of media carbohydrate on fermentation products. Acetic and propionic acids were the major 14C-labeled fermentation products. 14C-Carbohydrates were fermented to lactic acid and then to acetic and propionic acids. Distribution of radiolabeled fermentation products was effected by the broth carbohydrate and the time postinoculation.

A continuous-flow culture system was used to isolate and maintain a mixed culture of cecal bacteria from adult broilers composed of 29 bacterial strains representing 10 genera. Broiler chicks were treated with the mixed culture in the drinking water on the day of hatch and challenged orally with 10(4) Salmonella typhimurium 2 d after treatment. The experiment was repeated in four separate trials using newly hatched chicks. The concentration of propionic acid and total volatile fatty acid (VFA) in the cecal contents was determined 2 d after treatment and at 10 d of age. Compared with controls, the number of treated chicks that were cecal culture-positive for Salmonella decreased (P < .01) in each of the trials. Additionally, the number of Salmonella in the cecal contents of the treated chicks at 10 d of age was decreased (P < .005) compared with controls in each trial. The decreased number of Salmonella in the cecal contents of the 10-d-old treated chicks was correlated with elevated concentrations of propionic acid (P < .05) and total VFA (P < .1) in the cecal contents of the treated chicks 2 d after treatment. The results indicated that VFA-producing bacteria present in the mixed culture became rapidly established in the ceca of the treated chicks and effectively increased resistance to S. typhimurium challenge.

We previously reported that the prophylactic, intraperitoneal administration of supernatants from concanavalin A-stimulated T cells derived from Salmonella enteritidis (SE)-immune White Leghorn hens (i.e., SE-immune lymphokines or ILK), conferred protection to neonatal White Leghorn chicks against SE organ invasion. In the present study, we evaluated the effects of in ovo administration of ILK on hatchability, hatch weight, in vitro bactericidal activity of heterophils, and protection against SE organ invasion in neonatal White Leghorn chicks. On Day 18 of embryogenesis, injections were made into the amnion with either ILK or nonimmune ILK (NILK) or were not injected (untreated). On the day of hatch, whole blood was collected from 20 of the chicks per treatment group for heterophil isolation. All remaining chicks were orally challenged with 5 x 10(4) cfu SE. Twenty-four hours after SE challenge, organs (liver and spleen) from the chicks were cultured for SE. Hatchability of ILK- and NILK-treated chicks was not different from that of untreated chicks. Hatch weights of ILK-treated chicks were approximately 1 g less (P < .05) than that of NILK-treated or untreated chicks. In vitro bactericidal activity of peripheral blood heterophils derived from ILK-treated chicks was increased (P < .05) above activity from heterophils derived from NILK-treated or untreated chicks. Organ invasion with SE was markedly and significantly decreased in the ILK-treated chicks as compared with chicks treated with NILK or untreated chicks. These results suggest that in ovo administration of ILK confers protection to neonatal chicks against SE organ infectivity at hatch.

Drag-swab samples were collected from 18 poultry houses at 9 broiler farms. Fifty drag-swab samples were tested for Salmonella by microbiologic culture using selective enrichment and the polymerase chain reaction (PCR) with oligonucleotide primers specific for all members of the genus Salmonella. Drag-swab samples were tested for Salmonella using PCR before and after enrichment. Only one sample was positive by PCR prior to enrichment. Forty-seven of the drag-swabs samples tested after enrichment were positive for Salmonella using PCR, and 29 were positive by microbiologic culture. All but one of the culture-positive samples were positive by PCR; this discordant sample was classified as indeterminate by PCR. Salmonella was identified in houses from all nine farms by PCR and eight of nine farms by microbiologic culture. Salmonella was found in all 18 houses by PCR and in 15 of 18 houses by microbiologic culture. In this study, PCR was significantly (P < .001) more sensitive than culture for environmental monitoring of Salmonella using drag-swabs.

The effect of dietary lactose and of cell concentration of a continuous-flow (CF) derived bacterial culture on Salmonella typhimurium cecal colonization in 10-d-old broiler chickens was examined. One-day-old chicks were provided 1) no CF culture and the control diet; 2) no culture and a 2% lactose diet; 3) CF culture (10(8) or 10(11) anaerobic cfu) and control diet; or 4) CF culture (10(8) or 10(11) anaerobic cfu) and 2 to 4% lactose diet. All groups were challenged orally with 10(4) S. typhimurium at 3 d of age. Salmonella typhimurium growth in cecal contents was significantly decreased (P.05) at 10 d of age when 2% lactose was used in combination with CF culture containing 10(8) or 10(11) anaerobic cfu. The protection factor (log10 Salmonella control diet divided by log10 Salmonella treatment group) for these treatment groups was 2.49 and 9.26, respectively. Results indicate that birds treated with CF culture and maintained on 2% dietary lactose are protected against S. typhimurium colonization. Additionally, inoculating birds with CF culture containing a higher concentration of anaerobic colony-forming units resulted in a substantially higher protection factor.

Cecal anaerobic bacteria from adult broilers were cultured in media containing .25% glucose or .25% lactose. Media also contained either [14C]-labeled lactose, glucose, galactose, or lactic acid as metabolic tracers. Cultures were analyzed at 4, 8, and 12 h for pH, radiolabeled and unlabeled volatile fatty acids, and lactic acid. The pH values of cultures containing .25% lactose were significantly (P.05) higher than the pH values of cultures containing .25% glucose. Lactose cultures reached their lowest pH more slowly than glucose cultures. Concentrations of unlabeled volatile fatty acids increased and lactic acid decreased during incubation of the cultures. Radiolabeled sugars and lactic acid were more readily metabolized to volatile fatty acids in media containing lactose than in media containing glucose. The preferred metabolism of [14C]substrates, independent of media carbohydrate, was in the following order: lactic acidgalactose, lactoseglucose. The volatile fatty acids in which radiolabel was most concentrated were acetic acid, propionic acid, or butyric acid.

The effect of 14 or 19 days of 10% dietary lactose administration on Salmonella enteritidis (SE) colonization and histological, morphometric, and organic acid changes of the ceca were investigated. At Day 13 or 18, chicks were challenged with 10(8) cfu of SE. Chicks were killed and cultured 24 h later. A reduction in the total number of positive SE organ invasions was observed following 14 days (P < .001) or 19 days (P < .005) of treatment in chicks fed with lactose. Histological examination revealed a marked reduction in lamina propria thickness of ceca, as well as subjective epithelial cell proliferation from chicks following either 14 or 19 days of lactose administration. Using morphometric analysis, a reduction in the mean lamina propria thickness in chickens fed with lactose during 14 or 19 days was observed as compared with controls (P < .05). Yet, an increase (P < .05) in the mean epithelial cell length in both lactose-treated groups was observed as compared with controls. Lactose decreased luminal pH (P < .05) and increased the concentration of acetic, propionic, butyric, and lactic acid (P < .05). These data indicate that lactose-induced resistance to SE organ invasion is associated not only with an increase in organic acid concentration but also with measurable morphological changes of the cecal mucosa.

By conventional trayliner (hatcheries) and drag swab assembly (broiler houses) culture methods, the isolation distribution of Salmonella serotypes from five commercial broiler hatcheries (three sample times) and 13 broiler farms (eight sample times) was evaluated. A total of 11 different Salmonella serotypes were isolated from hatcheries, with Salmonella heidelberg (9/30) and Salmonella kentucky (6/30) accounting for 50% of the total isolations. Of 700 chick paperpad trayliners sampled, regardless of lot (breeder flock source) or hatchery, 12% were positive for Salmonella. When 10 individual trayliners were cultured from individual lots (same breeder flock source), Salmonella was detected in 24/57 lots (42%). Multiple serotypes were simultaneously isolated from the same lot on three occasions (6%). Of the 21 lots that were serially sampled, the Salmonella serotype detected was different within lots eight times (38%) on at least one occasion of two or more sampling times. Of the 196 individual broiler houses sampled, 44 were positive for Salmonella (42%). Twelve different serotypes were isolated from broiler houses during this study. The serotypes isolated most frequently were S. heidelberg (34/94) and S. kentucky (22/94). These two serotypes accounted for 59.6% (56/94) of the total broiler house isolations. Of the 38 houses that were serially sampled, two or more serotypes were detected in the same broiler house on 20 occasions (53%). Of the 38 serially sampled houses (four or more times), a consistent Salmonella serotype was detected in five houses (13%). In only 5 of the 38 (13%) serially sampled houses did we fail to detect Salmonella on four or more samplings. No significant difference in Salmonella isolation frequency was observed between poultry houses using new or used litter. These data support previous findings indicating that paratyphoid Salmonella serotypes are prevalent in some broiler hatcheries and houses. Further, the observation of multiple serotypes simultaneously and serially isolated from the same breeder hatchery lots suggests that breeder flocks may be infected with more than one serotype, possibly providing a source for multiple serotype infections in progeny grower flocks.

Broiler chicks were treated by oral gavage on the day of hatch with a continuous-flow competitive exclusion culture (PREEMPT). At 4 h, 1 day, or 2 days posttreatment, chicks were challenged by oral gavage with 10(2) or 10(4) Salmonella CFU to determine the effects of challenge time on Salmonella cecal colonization. Cecal propionic acid concentrations in two trials increased (P < or = 0.001) within 1 day posttreatment in chicks given PREEMPT, and the increases were indicative of the establishment of the PREEMPT bacteria. Salmonella cecal populations decreased (P < or = 0.001) on average 6 log10 units in these two trials in chicks challenged 4 h posttreatment with 10(4) Salmonella CFU. In a third trial propionic acid did not increase significantly until 2 days after treatment, and there was no decrease in Salmonella colonization when chicks were challenged at 4 h after treatment. However, there were decreases in that same trial when chicks were challenged at 1 and 2 days after treatment. The early establishment of PREEMPT followed by challenges with 10(2) and 10(4) Salmonella CFU resulted in 3% and 3%, respectively, of the ceca testing Salmonella-culture-positive, compared to 28% and 95%, respectively, culture-positive ceca in untreated chicks. The results from this study indicated that in most instances young broiler chicks can be protected against cecal colonization when challenged with 10(2) and 10(4) Salmonella CFU as early as 4 h posttreatment on the day of hatch with the PREEMPT bacteria.

In this study we tested the hypothesis that the frequency of Salmonella isolation from protective foot covers worn in individual broiler production houses would compare favorably to isolation rates obtained from conventional drag-swab methods. Salmonella was detected with equal frequency from protective foot covers and drag-swab assemblies on nine individual broiler farms over three separate sampling periods. Salmonella was detected in 13 of a total 27 individual samplings by culturing the protective foot covers, whereas positive detections occurred in 16 of a total 27 samplings when using the drag-swab method. Of the total number of houses identified as positive, these frequencies were unaltered when evaluated against the flock status of each farm at the time of sampling. Both methods were equally as likely to detect Salmonella when houses were either vacant (awaiting the placement of the next flock) or occupied (currently housing chickens of any age). In addition to highlighting the development of a potential new Salmonella monitoring technique, this study reinforces our current understanding regarding the importance of stringent biosecurity practices on poultry farms.

Hematopoietic colony stimulating factors (CSF) regulate the growth and development of phagocytic cell progenitors and also augment functional activation of phagocytes. Granulocyte-CSF (G-CSF) is the CSF that acts specifically upon granulocyte progenitor cells and mature granulocytes. We have shown that lymphokines (ILK) from T cells of birds immunized against Salmonella enteritidis (SE) induce a granulocytic (PMN) inflammatory response in chicks challenged with SE. This inflammatory response was characterized by: (a) a dramatic emigration of granulocytic cells from the bone marrow into the peripheral blood, (b) an enhancement of the biological functions of the circulating PMNs, and (c) a directed influx of these activated PMNs to the site of bacterial invasion. In the current study, we determined the presence of G-CSF in ILK by Western blot analysis using a goat polyclonal antihuman G-CSF antibody (Ab). Using this Ab, we then evaluated the role of G-CSF in the ILK-induced protective inflammatory response in chickens against SE. Pretreatment of ILK with the Ab totally abolished the colony-stimulating activity of the ILK. Furthermore, Ab treatment of ILK resulted in: (a) an elimination of the ILK-induced peripheral blood heterophilia with a dramatic inhibition of ILK-mediated protection against SE organ invasion and (b) an elimination of accumulation of inflammatory PMNs in the peritoneum with subsequent decrease in the survival rate of chicks challenged i.p. with SE. Taken together these studies demonstrate for the first time the contribution of G-CSF to avian PMN activation and the immunoprophylaxis of SE infection by ILK in neonatal chickens.

A carrier system for IL2 is needed in order to circumvent the toxicity associated with high dose interleukin 2 (IL2) administration and its rapid clearance from circulation. Erythrocytes (RBC) coated with recombinant interleukin 2 (rIL2) provide a means of delivering IL2 into the system in a continuous, low dose manner which in turn maintains a low, potentially non-toxic, IL2 concentration. Murine RBC coated with rIL2 (RBC-rIL2) induce cytotoxicity (21–31%) upon cytotoxic testing of spleens cells stimulated in vivo. Using the murine Meth A sarcoma model, the effectiveness of this RBC-rIL2 vehicle is demonstrated in vivo by a 84% reduction in tumor size as compared to the soluble rIL2 treated mice. Moreover, the RBC-rIL2 vehicle is able to induce tumoricidal cytotoxicity with very low rIL2 concentrations (about 10,000 I.U. of rIL2 per mouse). These results indicate that rIL2 retains its biological activity when bound to the RBC and therefore could prove useful as a therapeutic delivery system for cancer treatment.

Salmonella typhimurium is a significant hazard to consumer health that is carried asymptomatically in poultry gastrointestinal tracts. Nurmi cultures may prevent Salmonella colonization in young chicks, but the mechanism of competitive exclusion is unclear. Modeling Salmonella's metabolism in pure culture may allow for greater definition in choosing strains for Nurmi cultures. The growth rates and affinity constants of S. typhimurium growing in amino acid-limited conditions were determined in batch culture and compared to primary poultry isolates of cecal strains. Serine and NH4Cl were the best N sources for growth of all organisms tested in this study. The fermentation response of S. typhimurium was also monitored in continuous culture at a slow dilution rate of 0.021 h-1. S. typhimurium was found to adapt to VL media, with trends in protein disappearance, Yglucose, and Yprotein. This may show that amino acid or protein concentrations may be an integral component of the initial establishment of S. typhimurium in the cecum.

Broiler chicks were inoculated orally at 1 day of age with a continuous-flow (CF) culture of anaerobic cecal bacteria and challenged with 10(4) Salmonella typhimurium 48 hr (at 3 days old) after inoculation to determine the effect of the CF culture (CF3) on Salmonella crop colonization. Chicks were assigned to four groups: 1) untreated control chicks, 2) challenged at 3 days old with Salmonella, 3) inoculated at 1 day old (day-of-hatch) with CF3, and 4) inoculated at 1 day old with CF3 and challenged at 3 days old with Salmonella. Crop pH decreased significantly (P0.05) 24 hr after inoculation in chicks provided with CF3. The pH of crops at 24 hr from control chicks (group 1) was 5.4 and the pH of crops from inoculated chicks (group 3) was 4.7. Decreased pH was accompanied by a significant increase (P0.05) in corp lactic acid from approximately 0.1 mmol/ml in control chicks to about 0.2 mmol/ml in chicks given the culture. Salmonella crop colonization decreased (P0.05) 4 hr after challenge from a 2.6 log10 colony-forming units (cfu) in Salmonella-control (group 2) chicks to 0.6 log10 cfu in CF3-inoculated (group 4) chicks. Although at 4 and 8 hr after challenge, there were decreased (P0.05) numbers of crops testing culture-positive for Salmonella regardless of treatment, Salmonella colonization decreased (P0.05) in chicks inoculated with CF3 as compared with controls. The results indicated that CF3 can effectively reduce Salmonella crop colonization.

We have shown previously that increased resistance to Salmonella enteritidis (SE) organ infectivity in 1-day-old chicks was conferred by the immunoprophylactic administration of SE-immune lymphokines (SEILK). These lymphokines have been found to be present in the cell culture media of concanavalin A-stimulated splenic lymphocytes obtained from SE-immunized chickens. In the present study we evaluated whether turkeys also produced SEILK and whether these lymphokines could protect 1-day-old chicks and turkey poults against SE liver invasion. In addition, we tested the ability of our original chicken SEILK to reduce SE liver invasion in turkey poults. Day-of-hatch chicks and turkey poults were injected intraperitoneally with immune lymphokines of either chicken or turkey origin. One hour later the birds were challenged per os with SE, and 20 hours later their livers were examined by bacteriological methods for the presence of SE. We found that SEILK induced from the splenic lymphocytes of SE-immunized turkeys reduced SE liver invasion in both chicks and turkey poults. Conversely, we also determined that SEILK produced by chicken splenic lymphocytes conferred protection against invasion by SE in turkey poults. This research is the first report of the production of SEILK in turkeys and also the first report on the cross-species activity of these effector molecules in chickens and turkeys.

The consistency and persistence of isolation of specific serotypes of Salmonella were evaluated on a 31-farm broiler complex following four complete sampling periods. A total of 25 different serotypes were isolated, with multiple serotypes and were frequently isolated simultaneously from individual farms. When the serotypes recovered from individual farms were further examined, common serotypes were isolated from individual farms on only seven occasions. Furthermore, on only five occasions were multiple serotypes recovered from individual farms on consecutive samplings. A casual association of common serotype isolations among farms with similar flock placement dates was observed in one sampling period. These results indicate little predictability or consistency of Salmonella serotype isolation on individual farms over time.

The effect of treatment with a newly developed characterized continuous-flow (CCF) culture composed of 29 strains of cecal bacteria on salmonellae colonization was evaluated in commercially reared broiler chickens. Newly hatched chicks in three flocks were sprayed with CCF culture as they were placed in rearing houses and compared with untreated flocks on the same farm. Hatchery transport tray liners and rearing house feed, water, and little samples were cultured for the presence of salmonellae. Cecal samples were cultured after 3- and 6-wk growout. Skin-feather samples were cultured at 6-wk growout. Fifty-five percent of the transport tray liners were contaminated with salmonellae on the day of chick placement. At 3 wk, salmonellae serotypes present on the tray liners were widely distributed in the litter of the rearing houses. The results indicated that exposure to salmonellae occurred before culture treatment and continued in the rearing houses during the 6-wk growout period. Salmonellae cecal colonization was decreased (P < .05) in two of the treated flocks at 3 wk and diminished (P < .07) in the third treated flock compared with control flocks. At 6 wk, skin-feather contamination and cecal colonization were decreased (P < .05) in one of the treated flocks whereas no treatment effect occurred in two of the treated flocks compared with controls. The results clearly indicate the necessity of implementing integrated programs to control salmonellae in both the hatchery and rearing house environments. The CCF culture served to enhance salmonellae colonization resistance and may serve as a useful component of an integrated control program.

Investigations in our laboratories have indicated that when Salmonella enteritidis (SE)-immune lymphokines--supernatants from concanavalin-A-stimulated T cells derived from SE-immune adult chickens--were administered intraperitoneally to 1-day-old chicks before SE challenge, they conferred protection against SE organ invasion within 24 hr. This resistance mediated by SE-immune lymphokines was associated with a concomitant increase in peripheral blood polymorphonuclear leukocytes that peaked 4 hr after SE challenge. In the present study, we evaluated efficacy of SE-immune lymphokines in protecting chicks against SE organ invasion and alterations in peripheral blood polymorphonuclear leukocyte counts. Administration of SE-immune lymphokines to chicks either 30 min or 6 days before SE challenge caused a significant reduction in SE organ invasion. However, when SE-immune lymphokines were administered 2 days after SE challenge, there was no reduction in SE organ invasion. Both prophylactic (before SE challenge) and therapeutic (after SE challenge) administration of SE-immune lymphokines caused a significant increase in numbers of peripheral blood polymorphonuclear leukocytes. Results from these studies suggest that SE-immune lymphokines have potential value as an effective prophylactic but not as a therapeutic modulator of early resistance to SE organ invasion in neonatal leghorn chicks.

A rapid sample processing method has been developed to detect low numbers of Salmonella typhimurium in a chicken cecal microbial consortium. Using phoP-specific primers under stringent amplification conditions and gene probe analysis, fewer than 100 colony-forming units (CFUs) were detectable when pure cultures were employed. When the polymerase chain reaction assay was run on cecal contents from birds infected with S. typhimurium, only positive cecal samples containing as few as 700 CFUs reacted to the assay, and the negative bird samples reacted only when the samples were spiked with S. typhimurium cells. The method employed for sample processing is simple and provides a sensitive means of detecting S. typhimurium-specific sequences in trace amounts in the presence of mixed chicken cecal microbial populations.

The results of four independent complete drag-swab samplings of a 31-farm broiler-production complex are described. Samplings of vacant poultry farms (i.e., between flocks) and occupied farms (i.e., containing chickens) were completed in four 3-day intervals beginning in July 1992 and ending in May 1993. During the first two sampling periods, two drag-swab assemblies were dragged through each individual house and pooled into one sample to evaluate overall incidence of Salmonella detection among vacant and occupied houses. During the second two sampling periods, four drag-swab assemblies were dragged through houses to evaluate frequency of Salmonella detection on individual assemblies within each house. Over the four sampling periods, mean isolation frequencies were 42.2% for vacant poultry farms and 76.2% for occupied poultry farms. When four assemblies were dragged in vacant houses and cultured separately, the probability of detecting salmonellae contamination increased with increasing numbers of assemblies, indicating that multiple drag-swab assemblies should be used for sampling either vacant or occupied broiler houses for the presence of Salmonella. On vacant farms, 89.5% of the total isolations occurred in only one house of two-house farms; the remaining 10.5% of farms were positive in both houses. Salmonella isolation from occupied farms occurred in 54.2% of the samplings in only one house, whereas 45.8% of occupied farms were positive in both houses. These data show a higher frequency of Salmonella isolation on multiple swabs in occupied poultry houses, possibly indicating a quantitatively greater presence of Salmonella in these houses.

Doxorubicin was encapsulated in canine erythrocytes, treated with 0.32% glutaraldehyde, and administered at a dosage equivalent to 30 mg of free doxorubicin/m2 of body surface area to dogs with diagnosis of lymphosarcoma. Compared with administration of free doxorubicin, this method of drug delivery substantially reduced peak plasma concentration and prolonged higher plasma concentration of doxorubicin. As such, this method was comparable to continuous IV infusion. Previous studies have indicated this method's potential for reduction in toxic side effects, particularly cardiotoxicosis, while allowing higher total doses of doxorubicin to be administered. In this study, doxorubicin encapsulated in glutaraldehyde-treated erythrocytes induced a triphasic exponential decay of doxorubicin from plasma, the highest relative contribution to the total area of the curve being the terminal phase. The treatment was effective in inducing complete and partial remissions of lymphosarcoma, with minimal acute toxicosis and no evidence of cardiotoxicosis. However, substantial, unanticipated, chronic, nonregenerative myelosuppression developed, and was mot strikingly expressed as profound thrombocytopenia. Efforts to ameliorate or circumvent this toxic effect will be required prior to further consideration of this doxorubicin delivery system for treatment of systemic neoplasia.

Mouse red blood cells (RBC) were incubated with recombinant human interleukin 2 (rIL 2) in protein-free media. Of the added cytokine, about 20% was bound to the cells. Dissociation of rIL 2 from RBC commenced when the carrier cells were transferred into fresh media. The release from the RBC vehicle was enhanced in the presence of soluble protein. When rIL 2-coated RBC (RBC-IL2) were incubated in protein-containing media for 24 h to release the majority of bound rIL 2, and then were subsequently incubated in fresh media with an IL 2-dependent cytotoxic T cell line (CTLL), residual bound rIL 2 was still released as demonstrated by CTLL proliferation. RBC were covalently coupled to specific monoclonal antibody (mAb) towards the lymphocyte cell-surface marker Thyl.2, coated with rIL 2, subsequently exposed to the target CTLL cells, then evaluated for CTLL/RBC rosette formation. The attachment of mAb to the RBCs surface did not markedly change the succeeding rIL 2 adsorption, and the bound rIL 2 did not impair antigen recognition by the mAb. At higher rIL 2 concentrations (1200-1500 i.u./10(8) cells), RBC-IL2 plus Thy1.2 mAb provided higher CTLL response than when RBC-IL2 plus non-specific mAb was used thus demonstrating enhancement by targeting. However, the targeting effects were not seen with lower rIL 2 concentrations (80-110 i.u./10(8) cells).

Leghorn chicks were treated with cultures of cecal bacteria from adult chickens by crop gavage, upper body spray, or vent lip application on the day of hatch. The chicks were challenged orally with 10(4) Salmonella enteritidis (SE) at 3 d of age and evaluated for SE cecal colonization at 10 d of age. The concentration of volatile fatty acids (VFA) in the cecal contents was determined on the day after culture treatment and at 10 d of age. Compared with controls, SE colonization was significantly decreased in each of the treatment groups. Vent lip application of a single .05-mL drop of cecal bacteria culture resulted in resistance against SE challenge comparable to crop gavage or spray treatment with .5 mL of culture. Resistance to SE challenge was directly associated with the concentrations of total VFA and propionic acid in the cecal contents of the treated chicks on the day after culture treatment. The results indicated that cecal bacteria from adult chickens that increase SE colonization resistance may rapidly become established in the ceca of newly hatched chicks following contact with the vent lips.

The protective effect of cecal bacteria cultures on Salmonella enteritidis cecal colonization was evaluated. Competitive-exclusion cultures were administered by crop gavage, in first drinking water, by whole body spray, or encapsulated in alginate beads and provided in feed pans. Leghorn chicks were treated with cultures of cecal bacteria on the day of hatch and challenged orally with 10(4) S. enteritidis 2 days after treatment. Salmonella cecal colonization was evaluated 7 days after challenge. No Salmonella organisms were detected in the ceca of chicks treated with cecal cultures by crop gavage. Chicks treated with cecal cultures in the drinking water or by spray application showed comparable protection and significant decreases (P0.05) in the number of Salmonella in the cecal contents compared with untreated controls. The consumption of cecal bacteria encapsulated in alginate beads significantly decreased (P0.05) Salmonella cecal colonization compared with control treatment, but it provided less protection than the other treatment methods evaluated.

The effect of incubating Salmonella typhimurium and S. typhimurium cell-free extracts with isolated intestinal segments from 1-day-old chicks was evaluated using scanning electron microscopy. Incubation of segments with intact bacteria or with cell-free extract resulted in the loss of mucosal epithelial integrity after as little as 30 min incubation. Loss of mucosal epithelial integrity was evidenced by the complete shedding of the epithelium. The addition of 2.5% D-mannose to the incubation medium inhibited the loss of epithelial cells, whether intestinal segments were incubated with intact bacteria or with cell-free extract. These results indicate that S. typhimurium exerts a D-mannose-sensitive cytotoxic effect on the mucosal epithelium of isolated intestinal segments and that the cytotoxic effector is present in cell-free extracts of the bacteria.

This study evaluated the effect of Eimeria tenella infection on Salmonella typhimurium colonization of broiler chicks following oral inoculation with anaerobic cecal flora either alone or with a treatment of 10% dietary lactose. The number of chicks colonized by S. typhimurium was significantly lower (P0.05) in groups inoculated with cecal flora with or without the dietary lactose treatment than in control groups. In addition, significantly fewer S. typhimurium organisms were isolated from the cecal contents of chicks inoculated with anaerobic cecal flora and treated with 10% dietary lactose than in the control chicks. Infection with E. tenella had no effect on the resistance of treated groups to S. typhimurium colonization of the ceca but did cause higher S. typhimurium colonization in the untreated control chicks. The results indicated that: A) anaerobic cecal flora given orally on the day of hatch to chicks with or without dietary lactose enhanced the resistance of the birds to Salmonella cecal colonization; and B) coccidial infection had no detrimental effect on this resistance to Salmonella colonization.

Cytokines, derived from either concanavalin A-stimulated Salmonella enteritidis-immune chicken T lymphocytes [SE-immune Lymphocyte Stimulated Soluble Factor (LSSF)] or lipopolysaccharide-stimulated SE-immune chicken macrophages [SE-immune Macrophage Stimulated Soluble Factor (MSSF)], were evaluated for their ability to increase resistance to SE organ invasion in day-old Leghorn chicks. In Trial 1, day of hatch chicks were injected i.p. with either SE-immune LSSF or Se-nonimmune LSSF (control). In Trial 2, chicks were similarly injected with either SE-immune MSSF, SE-nonimmune MSSF, or SE-immune LSSF (positive control). Thirty minutes postinjection, all chicks were gavaged with an invasive dose of SE. Twenty-four hours later, livers and spleens from all chicks were cultured for SE. In Trial 1, SE-immune LSSF caused a rapid and marked protection (P < .01) against SE infection as determined by the number of chicks that were culture positive regardless of challenge dose. In Trial 2, SE-immune MSSF was not associated with protection against SE organ infection. These experiments demonstrate that SE-immune LSSF, but not MSSF, are able to confer protection against SE organ invasion in day-old Leghorn chicks. Thus, it appears that the stimulated immune T cell, and not the macrophage, is responsible for producing the soluble products that protected the chicks.

Newly hatched broiler chicks were provided a corn/soybean meal-based ration treated with propionic acid at 30 mumol/g of feed ration. At 3 days of age, the chicks were challenged orally with 10(4) Salmonella typhimurium. Crop contents from 4-day-old chicks that were provided dietary propionic acid contained significantly higher concentrations of propionic acid (4.0 to 6.8 mumol/g crop contents) than crops from challenged control chicks provided untreated feed (0.9 to 1.5 mumol/g crop contents). Provision of dietary propionic acid on feed as a dry powder in five trials or a liquid application in three trials had no significant effect on crop or cecal pH. Significant decreases in Salmonella in the crop and ceca were detected in one trial, but the decreases were likely the result of the presence of anti-salmonellae bacteria rather than the dietary propionic acid. Results indicate that propionic acid in the feed was ineffective in reducing Salmonella infection in the crop and ceca.

A defined bacterial culture protective against Salmonella typhimurium cecal colonization in broiler chicks was derived utilizing a continuous-flow (CF) culture apparatus. Chicks receiving the CF culture in combination with a diet containing dietary lactose were protected against cecal colonization by S. typhimurium. The culture consisted of a mixture of gram-positive and gram-negative facultative and strictly anaerobic bacteria. The isolates were identified as Enterococcus avium, two strains of Enterococcus faecalis (designated A and B), Lactococcus lactis, Lactobacillus animalis, a Lactobacillus that could not be identified to species level (designated strain CMS), Citrobacter freundii, Escherichia coli, E. fergusonii, Bifidobacterium animals, and Propionibacterium acidipropionici. Results indicated that CF cultures can be used as a tool to identify bacteria which are antagonistic to S. typhimurium in the chick cecum.

Mouse and human erythrocytes are inherently different with respect to slow dialysis encapsulation used in preparing carrier erythrocytes. Fluorescein isothiocyanate (FITC)-dextran was added to five different stages of the encapsulation process to discern when endocytic vesiculation occurred. Mouse erythrocytes were much more unstable than human cells, with as many as 50% of mouse cells showing vesicles, as determined by flow cytometry. Mouse erythrocytes showed the ability to form vesicles at each stage beyond the washed-packed-cell stage. Up to 20% of the human cells formed vesicles at stages beyond the annealed-cell stage. Although vesiculation can occur at any stage of the encapsulation process, with the exception of washed-packed cells, the actual amount of FITC-dextran incorporated in the cells is extremely low when compared with dialysis encapsulation, namely 50 ng/10(7) cells as against 2,000 ng/10(7) cells. Thus preparation of carrier erythrocytes by slow hypo-osmotic dialysis induces certain instabilities that lead to a substantial percentage of cells with endocytic vesicles, while the actual amount endocytosed is low. The differences in vesiculation observed between human and mouse erythrocytes is apparently related to the intrinsic properties of the cells and is consistent with the fact that mouse erythrocytes are more fragile when undergoing slow dialysis than are human erythrocytes.

An in vitro continuous-flow (CF) culture system was utilized to isolate and maintain a defined mixed culture of indigenous cecal bacteria from adult broilers. The protective effects of the defined CF culture and dietary lactose on Salmonella typhimurium colonization were evaluated in broiler chicks. The CF culture was administered to chicks by crop gavage on the day of hatch. Lactose was provided as 5% (wt/wt) of the feed ration. The chicks were challenged orally with 10(4) S. typhimurium at 3 days of age and evaluated for Salmonella colonization 7 days after challenge. The experiment was repeated in six separate trials using newly hatched chicks and CF culture that was maintained in continuous steady-state conditions from 42 to 190 days. Compared with controls, the mean number of S. typhimurium in the cecal contents of the chicks given CF culture and dietary lactose decreased significantly (P < .01) by 4.2 log10 units. Similarly, the numbers of Salmonella cecal culture-positive chicks was significantly decreased (P < .01) by 55% in the chicks given CF culture and lactose. The results indicated that a defined culture of indigenous cecal bacteria isolated and maintained in CF culture, together with dietary lactose, effectively controlled S. typhimurium cecal colonization in newly hatched broiler chicks.

Mixed cecal microflora obtained from a mature chicken were maintained in vitro in continuous-flow (CF) culture. The effect of the CF culture and dietary lactose on Salmonella typhimurium cecal colonization in broiler chicks was evaluated. When averaged across four replicates, chicks treated with the culture alone (1.75 log10 decrease) or with 5% dietary lactose alone (2.98 log10 decrease) were protected against S. typhimurium. Optimum protection against S. typhimurium was observed when birds were treated with the culture in combination with dietary lactose (4.27 log10 decrease). Dietary lactose resulted in reduced cecal pH. A large increase in cecal propionic acid was observed in the birds given the CF culture. A significant correlation (P0.001) was observed between the cecal concentration of undissociated propionic acid and protection against S. typhimurium colonization (r = -0.78). The results indicated that indigenous cecal flora that protect against Salmonella colonization can be maintained without loss of efficacy in CF culture.

The toxic effects of mature, seed-bearing flatpea (Lathyrus sylvestris L cv Lathco) hay on sheep was studied in a feeding trial with 25 adult ewes. Five ewes were barren; 20 were in the last 10 w of pregnancy. The ewes were blocked by weight and reproductive status for assignment to treatment groups. Four groups of 5 ewes each were allotted to 1 of the following diets: 0% (controls), 35.0%, 52.5% or 70.0% flatpea (FP); and 1 group of 5 ewes was allotted to a diet of increasing levels of 17.5, 35.0, 52.5 and 70.0% FP. Flatpea hay was harvested in the mature, seed-bearing stage, pelleted, reground and incorporated into a basal (control) diet of 30% corn + 70% alfalfa by substitution of an appropriate amount of FP for alfalfa. The resulting diets were fed for up to 42 days. The toxic nature of FP was expressed by the following dose-related responses: Fluctuating reductions in feed intake upon initiation of diets containing 35% FP and above; occurrence of a potentially fatal, acute central nervous system syndrome at FP levels as low as 35%; and reduced weight gain at FP levels of 52.5% and above. A wasting condition was observed in 1 ewe fed 52.5% FP. Four of 5 ewes and 1 lamb fed increasing levels of FP developed acute poisoning. One of these ewes developed acute neurotoxicosis when consuming 52.5% FP; 3 other ewes and 1 40-d-old orphaned lamb developed neurotoxicosis upon raising the FP level to 70.0%.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of 14 or 19 days of dietary capsaicin (18 ppm) on Salmonella enteritidis infection and histological, morphometric, and pH changes of the ceca was investigated. At day 13 or day 18, chicks were challenged with 10(8) colony-forming units of S. enteritidis. Chicks were killed and cultured 24 hours later. The total number of S. enteritidis-organ-culture-positive chicks was significantly lower among chicks fed capsaicin for either 14 or 19 days than among controls (P0.05). Subjective histological examination revealed a mild to moderate infiltration of mononuclear cells and heterophils in lamina propria of ceca, as well as epithelial cell proliferation in chicks following either 14 or 19 days of capsaicin administration. Using morphometric analysis, the mean lamina propria thickness and mean epithelial cell thickness in chickens fed capsaicin for 14 or 19 days were significantly greater than in controls (P0.05). Capsaicin significantly decreased luminal pH in both trials (P0.05). These data indicate that the observed capsaicin-induced resistance to S. enteritidis organ invasion is associated with measurable pH and morphological changes of the cecal mucosa.

Broiler chickens were inoculated orally with 10(6) Salmonella typhimurium on the day of hatch. Twenty-four to 72 hr after challenge, the chicks were inoculated orally with cecal microflora that had been repeatedly passed through lactose-fed broiler chicks. In vivo passage proved to be a convenient and practical method for preserving protective anaerobic flora. These organisms effectively reduced S. typhimurium concentrations in the cecal contents by 4-5 orders of magnitude, even when given 24 to 72 hr after Salmonella challenge inoculation.

The effect of dietary administration of lactose and used poultry litter, containing cecal and fecal droppings from adult broilers, was evaluated for protective effects against Salmonella enteritidis colonization in leghorn chicks and 16-week-old hens. The addition of used litter as 5% of the feed ration significantly decreased (P0.01) Salmonella cecal and organ colonization in the chicks. Provision of used litter or used litter and lactose in the feed failed to provide protection against Salmonella colonization in the hens. The results indicated that resistance to S. enteritidis colonization may be effectively increased in leghorn chicks by exposure to adult intestinal flora present in used litter. Furthermore, the results suggest that microbiological strategies employing adult intestinal microflora that increase Salmonella colonization resistance in young chicks may be ineffective in older hens.

The effect of used pine-shaving litter from broiler floor pens on Salmonella colonization resistance was evaluated in broiler chicks. One-day-old chicks were placed in floor pens on fresh unused litter or on used litter. All chicks were challenged orally with 10(4) S. typhimurium at 3 days of age. The study was replicated in three trials with used litter that was collected and stored for 1 day (Trial 1), 4 days (Trial 2), or 50 days (Trial 3) before the start of each trial. Cecal concentrations of volatile fatty acids (VFAs) were significantly higher (P0.05) in chicks placed on used litter than in chicks on new litter. In all three trials, the number of Salmonella in the cecal contents and the number of Salmonella cecal-culture-positive chicks was significantly lower (P0.01) at 10 days and 20 days of age in the chicks on used litter than in the chicks on new litter. The results indicated that newly hatched chicks reared on used litter had higher cecal VFA concentrations and higher resistance to Salmonella colonization than chicks reared on new litter.

A method was explored to develop a general means to target erythrocytes to T-cells in vitro. Mouse erythrocytes were coupled with an anti-Thy-1.2 monoclonal antibody by two methods. Chromic chloride coupling of antibody was preferred to biotinylation. The morphology, osmotic fragility, and the hematological values of treated cells were normal compared with those of control erythrocytes. Antibody-coupled erythrocytes were incubated with cytotoxic T-lymphocytes (CTLL) in vitro at a 20:1 ratio. Approximately 60-70% of the CTLL formed rosettes. The cell mixture was subjected to gradient centrifugation and separated into four fractions. THe rosettes were clearly identified only in the treated group containing anti-Thy-1.2-coupled erythrocytes. No rosettes were found when aspecific monoclonal antibody was coupled to erythrocytes. Examination by scanning and transmission electron microscopy revealed CTLL with 4-5 erythrocytes attached to them but did not show any evidence of membrane fusion. Rosettes of CTLL incubated in vitro proliferated as well as CTLL alone and maintained their dependency on interleukin-2. Targeting of erythrocyte carriers to lymphocytes offers the potential for delivery of molecules directly to the target cell.