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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.

Canine erythrocytes were loaded with the antineoplastic drug doxorubicin and then treated with 0.16% glutaraldehyde. This procedure has been previously shown to slow down the efflux of doxorubicin from erythrocytes and to result in the selective targeting of the carrier erythrocytes to liver. Three dogs were treated each with 2 different schedules of IV bolus administration of doxorubicin (0.4 mg/kg of body weight): free drug and doxorubicin encapsulated in glutaraldehyde-treated erythrocytes. The 2 treatments yielded consistent differences in the plasma pharmacokinetic properties of doxorubicin and of its only metabolite, doxorubicinol. A triphasic exponential decay of doxorubicin plasma concentrations was observed on injection of the free drug. Conversely, in the case of erythrocyte-encapsulated doxorubicin, 4 phases of plasma concentrations of doxorubicinol were found. The plasma concentrations of doxorubicinol, after a steady increase during the first hour, followed patterns of decay comparable to those of the parent drug. On the basis of the kinetic variables calculated with the 2 administration schedules, area under curve concentrations of plasma doxorubicin were 136 micrograms.h/L (free infusion) and 734 micrograms.h/L (erythrocyte-encapsulated drug). Significant alterations of hematologic and hematochemical factors were not observed in the 3 dogs during and after the 2 treatments. On the basis of our findings, doxorubicin-loaded and glutaraldehyde-treated erythrocytes may potentially be used in the treatment of systemic and hepatic tumors in dogs.

Inclusion of lactose in the diets of chickens has been determined to reduce cecal colonization with Salmonella typhimurium. We hypothesized, therefore, that dietary lactose may be a practical means for reducing the prevalence of Salmonella contamination of chicken products. Because some strains of Salmonella are atypical and ferment lactose, we investigated the effects of dietary lactose on cecal colonization with lactose-fermenting S typhimurium. Broiler chicks were inoculated intracloacally with Lac+ S typhimurium selected for resistance to novobiocin and rifampicin. The chicks also were inoculated orally with certain anaerobes that do not effectively inhibit colonization by S typhimurium, but do appear essential for lactose mediated inhibition of cecal colonization. Control chicks were not given dietary lactose, and chicks in the experimental group were fed a diet containing 7% lactose. Enumeration of Lac+ S typhimurium in cecal contents revealed dietary lactose to be effective at controlling this organism. Control was correlated with changes in cecal pH and increases in undissociated volatile fatty acids, especially propionic acid.

A skin test to assess T-cell mediated delayed hypersensitivity (DH) and cutaneous basophil hypersensitivity (CBH) was evaluated in the interdigital skin of young chickens. Three-day-old chickens were sensitized with Mycobacterium tuberculosis, and the DH reaction was elicited in the interdigital skin in 10-, 17-, 24-, and 31-day-old chickens by intradermal injection of tuberculin. Cutaneous basophil hypersensitivity was elicited in the interdigital skin of 10- and 14-day-old chickens by a single intradermal injection of phytohemagglutinin-P (200 micrograms). The effect of immunosuppression on the results of interdigital skin test for DH and for CBH was evaluated in chickens that were treated with dexamethasone daily for 4 days before testing. The DH reaction, as indicated by a significant (P less than 0.01) increase in the mean interdigital skin thickness, was detectable in 10-day-old chickens and was consistently evident in 17-, 24-, and 31-day-old chickens. The DH response in the interdigital skin of 24-day-old chickens was comparable with that elicited in the standard wattle test. The CBH reaction, as indicted by a significant increase (P less than 0.005) in skin thickness, was evident in the interdigital skin of 10- and 14-day-old chickens. Treatment with dexamethasone significantly decreased (P less than 0.01) the DH and CBH reactions. Results of the study indicated that the interdigital skin test may be used to evaluate normal and suppressed cell-mediated DH and CBH reactions in chickens as young as 10 and 14 days old.

Fecal samples were collected from 200 feeder-calves on farms in Tennessee, after assembly at a Tennessee auction market, and after transport to a Texas feedyard. A final fecal sample was collected from each calf after 30 days of feedyard confinement. The fecal samples were cultured for the presence of Salmonella spp. Salmonella isolates were serotyped and antimicrobial drug-resistance patterns determined. The number of calves fecal culture-positive for Salmonella spp increased from 0 on the Tennessee farms and auction market to 3/200 (1.5%) at entry into the Texas feedyard, and 16/200 (8%) after 30 days of feedyard confinement. Salmonella serotypes isolated and the number of isolates of each serotype were S reading (8), S cerro (4), S newbrunswick (3), S anatum (2), and S typhimurium (copenhagen; 2). All Salmonella isolates were resistant to 5 or more of 13 antimicrobial drugs tested. Salmonella reading isolates were resistant to 10 or 11 of 13 antimicrobial drugs. The results indicated that the calves could have been infected with Salmonella spp prior to or during the course of the study, and that marketing stress as they moved from farm through feedyard may have induced fecal excretion of salmonellae. In addition, the pattern of antimicrobial drug resistance in the Salmonella isolates was broad.

Carrier erythrocyte survival was investigated in intact and splenectomized calves. Survival curves for both were biphasic, with a characteristic early loss of 10% to 15% of the carrier cells in intact calves followed by a half-life of 22 to 24 days. In splenectomized calves, 50% to 75% of the carrier cells disappeared from circulation in the first 24 hours. The 2nd phase of survival showed a 44-day half-life for the remaining carrier cells. Erythrocytes from splenectomized calves appeared to be permeable to [14C]sucrose and were more susceptible to irreversible hemolysis from the dialysis encapsulation procedure. Blood volume determination showed a 22% decrease after splenectomy.

Canine carrier erythrocytes are prepared by dialysis to contain [14C]-sucrose. The procedure allows over 37% of the available sucrose to be encapsulated in the erythrocytes. Cell recovery from the dialysis procedure is greater than 80%. Carrier erythrocytes were prepared from 4 breeds of dogs in less than 2 hours. Although there were some animal-to-animal differences, breed differences were not noticeable in carrier cell preparation. The carrier or resealed erythrocytes contain ca 70% of their hemoglobin and when injected circulate with a half-life of 36 days. These carrier cells could thus serve as a drug delivery vehicle to provide circulating concentrations of encapsulated drug.

Porcine carrier erythrocyte survival was measured in adult pigs. [14C]Sucrose-loaded erythrocytes had a biphasic survival curve, with as much as 50% of the cells removed from circulation in the first 24 hours. The remaining cells had a 35-day half-life. Encapsulation values were measured for porcine erythrocytes and entrapment of [14C]sucrose was greater than 45%. Addition of inosine and glucose to the dialyzed cells and to the final wash buffer before reinjection of autologous cells did not improve their survival.

Tetracycline was encapsulated in erythrocytes with a dialysis technique. The effect of drug concentration (0.2 mg/ml of erythrocytes) on encapsulation of [14C]sucrose and [3H]tetracycline was reduction of the tetracycline encapsulation, but not [14C]sucrose. Carrier erythrocytes containing tetracycline reinjected in calves were studied for their pharmacokinetic constants. The drug half-life was 6.7 hours, with an overall elimination constant of 0.104 hour-1.

Chemotherapeutic doses of methotrexate (MTX) were encapsulated in as little as 1 ml of canine carrier erythrocytes. Carrier erythrocytes prepared by a dialysis technique contained more than 3.0 mg of MTX/ml of cells. In vivo pharmacokinetic studies indicated that most of the drug leaked from the circulating MTX-loaded erythrocytes. In dogs, glutaraldehyde treatment of MTX-loaded erythrocytes prevented drug leakage and targeted more than 50% of the drug to the liver.

The human anti-cancer drug Cytosine arabinoside (ana-C) was encapsulated in canine erythrocytes. When these autologous carrier cells were injected into dogs, the drug was slowly released into plasma. Erythrocyte encapsulation increased the plasma half-life of ara-C in dogs about 50-fold over that of the injected free drug. The whole blood half-life of encapsulated drug was 4.6 days and the elimination constant was 0.150 day-1. Erythrocyte encapsulation holds promise as a drug-delivering technique for treatment of some human and animal diseases, particularly with drugs that are highly toxic or have normally short biological half-life. It also could be used to sustain given concentrations of drugs.

Calves were inoculated with helminths to induce a severe anemia. Increased erythrocyte mean cell volume caused by acute parasitism made erythrocytes more resistant to osmotic stress as reflected in the percentage of encapsulation parameters. Preparation of resealed carrier erythrocytes before and during parasitosis was studied. Low percentage of encapsulation of the marker [14C]sucrose could be corrected by extended dialysis time. Typically, 40% encapsulation was found in erythrocytes from healthy calves, whereas only 20% encapsulation was found for erythrocytes from helminth-infected animals.

Bovine carrier RBC containing [14C]sucrose as a marker were stored for 23 days in protein-free media. Cell recovery was highest with a phosphate buffer (pH 7.0) containing 2 mM MgCl2, 5 mM inosine, 5 mM adenosine, and 10 mM glucose. A cell recovery of 60% was observed after 23 days of storage at 4 C. Stored carrier erythrocytes reinjected into cattle had an 18-day half-life, which is less than that for fresh carrier RBC.

Small and large molecules were encapsulated in bovine RBC by a dialysis process. Encapsulation efficiencies were approximate 30%, yielding normal carrier cells. The new process of encapsulation used hollow fibers and was a continuous-flow system. Preparation of carrier RBC by hollow-fiber dialysis was compatible with blood banking principles. The continuous-flow system was easily assembled from commercially available hollow-fiber units.