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A fusion protein of β-galactosidase and Eimeria tenella produced in a recombinant Escherichia coli strain was injected into chickens and elicited partial protection against an oral challenge with Eim. tenella parasites. The fusion protein contained a 31 kilodalton (kD) coccidial antigen designated as 5401. The DNA sequencing of the 5401 antigen-coding sequence revealed that this protein segment was highly negatively charged and strongly hydrophilic, and contained an amino-acid sequence repeated five times. A dose-titration study showed that immunizing chickens with a single subcutaneous injection of the 5401 antigen at 1,200 to 4,800 nanograms (ng)/bird in Freund's complete adjuvant decreased lesion scores, mortality, and feed conversions compared to unimmunized, challenged controls. Using the 1,200 and 2,400 ng/bird of the 5401 antigen, group weight gains were higher than for the unimmunized, challenged birds. In three other trials using the 5401 antigen at 2,400 ng/bird with light, medium, and heavy coccidial infections, significant protection was evidenced by reduced lesion scores, increased individual weight gains, or both. In addition, feed conversions were reduced when compared with unimmunized controls or birds immunized with a noncoccidial protein E. coli extract. Western blot analysis of sporozoite preparations with serum from 5401-immunized birds labeled two antigenic bands of 66 and less than 200 kD. These results indicate that the coccidial proteins produced in E. coli are potentially effective immunogens for protecting chickens against avian coccidiosis.

We have measured lipolytic activity in gastric aspirates obtained at birth in a group of 142 infants. The infants ranged in gestational age from 26 to 41 wk. Lipolytic activity, measured by the hydrolysis of long chain triglyceride ([tri-(3)H]oleate), and expressed as nanomoles FFA per milliliter gastric aspirate per minute was 333+/-66 in 55 small premature infants (gestational age 26-34 wk and body wt 750-2,000 g) and 558+/-45 in a group of 87 larger infants (gestational age 35-41 wk and body wt 2,020-4,000 g). No activity was detected in seven infants with an unusually low pH in the gastric aspirate, 2.88+/-0.44 (compared with a mean pH level of 5.59+/-0.22 in the other 135 infants). Attempts to characterize this lipase showed that it has a molecular weight of 44-48,000, pH optimum of 3.0-5.0, that FFA acceptors (albumin) stimulate activity, whereas bile salts, taurocholate and glycocholate, cause marked inhibition at concentration3 mM. Our survey shows that enzyme activity is present as early as 26 wk of gestation, increases with gestational age, and has the same characteristics throughout gestation. The data show that the lipase in gastric aspirates differs from pancreatic lipase, but closely resembles human and rat lingual lipase. Because the lipase has a low pH optimum and does not require bile salts, it can act in the stomach where it initiates the hydrolysis of dietary fat. We suggest that intragastric lipolysis is probably of major importance in the newborn and especially in the premature infant where it compensates not only for low pancreatic lipase, but in addition, helps to overcome the temporary bile salt deficiency through the formation of amphiphilic reaction products.

A cDNA clone derived from sporulated oocysts of Eimeria tenella and encoding the expression product GX3262 was identified using a monoclonal antibody raised against Eimeria acervulina sporozoites. The cDNA fragment containing the coccidial antigen gene was cloned in bacteriophage lambda gt11, transferred to a plasmid, and introduced into Escherichia coli for analysis of the gene products. The strain carrying the plasmid produced GX3262 as part of a fusion protein consisting of the first 1,006 amino acids of E. coli beta-galactosidase and 112 amino acids of the E. tenella protein of approximately 12 kilodaltons. Partially purified antigen, heat-killed recombinant bacterin, and live E. coli containing the recombinant coccidial antigen were used to immunize 1-week-old or newly hatched broiler chicks. Several immunization protocols were utilized, including boosts with partially purified beta-galactosidase-GX3262, bacterin, or small numbers of live E. tenella oocysts. After challenge with an experimental E. tenella infection, the birds were evaluated by scoring cecal lesions to determine the level of protection. The greatest degree of protection was seen after only a single immunization of 2-day-old birds with a live recombinant E. coli preparation. The results presented here identify GX3262 as a potential candidate coccidial vaccine antigen and provide evidence for the first time that newly hatched chickens can be successfully vaccinated with a recombinant antigen.