Search

In order to demonstrate the population dynamics of the northern fowl mite (NFM), Ornithonyssus sylviarum (Canestrini and Fanzago), and to characterize the chicken’s immune response to the mites, White Leghorn hens were infested with mites. Visual estimates revealed populations peaked on Wk 5, 4, and 3 after being infested with 10, 50, or about 2,000 mites, respectively. Individual feathers pulled from the vent area of hens 4 wk (28 days) after the hens were infested with either 10 or 50 mites had means of 7,513 and 7,009 mites, respectively. Estimated mean total populations of NFM on these same hens were 182,000 and 258,000, respectively. In two replications, actual counts of viable NFM following infestation with 50 mites increased ca. 10-fold by the 2nd wk after infestations were established in the feathers and then another two-fold during the 3rd wk. Populations in the 4th wk were about one-third of levels observed during the 3rd wk and continued to decline rapidly. Western blot analyses demonstrated the appearance of a mite-specific antibody in sera of White Leghorn hens that was approximately proportional in time of appearance and intensity to estimated NFM populations. Detection of mite-specific antibodies in the hens’ sera continued through 12 wk of the study even though mite populations declined after 3 to 6 wk. The rapid increase and then decline of NFM following infestation in previously uninfested chickens and the fact that the immune response persisted strongly suggests that the population decline was due to an immune response. Therefore control of the mite population by a vaccine may be possible.

The contamination potential of sterile disposable plastic syringes was evaluated after subjecting the syringes to both simulated in-use conditions and an intentional microbial challenge. Lots of 20 Luer-lock syringes in 10 or 12-cu cm and 20- or 30-cu cm sizes from three manufacturers were tested. Sampling was conducted using 30-ml vials of sterile aerobic culture media containing 14C-labeled substrates. Microbial contamination was confirmed by both visual observation of the turbidity caused by colonization and instrumental detection of 14CO2 caused by the microbial metabolism of 14C-substrates. No contamination of 120 samples was found after the ribbed plunger shaft was grasped by a bare, unprepared dry hand during five cycles of filling and injecting the medium into the vials without removing the needles from the stoppers. When this sampling technique was applied to the syringes inoculated on the upper piston surface with Bacillus subtilis suspension, a 100% contamination rate was observed in 120 samples each under both positive and negative in-vial pressure. Grasping the ribbed plunger shaft of disposable plastic syringes with a dry bare hand did not compromise the sterility of the syringe contents in this study; however, this practice should be avoided when possible. Personnel should absolutely avoid introducing fluid-borne microbial contaminants into the distal barrel end of these syringes because the contents are readily labile to contamination under these conditions.

Good morning, ladies and gentlemen, and welcome to “Biotechnology for Solving Agricultural Problems,” the tenth in the series of Beltsville Agricultural Research Center Symposia.

Weight and biochemical studies were conducted on 2-week-old turkeys inoculated with 10(4) to 5 X 10(5) sporulated Eimeria meleagrimitis oocysts, on their pair-fed controls (equivalent food intake), and on control turkeys fed ad libitum. Food consumption and rate of weight gain of all inoculated and pair-fed turkeys fell sharply on day 4 postinoculation (PI), but deaths occurred primarily among the birds inoculated with 5 X 10(5) oocysts. Heart weights (expressed as percentage of body weight) were reduced in inoculated and pair-fed birds, but liver, spleen, and pancreas weights did not differ from those of either control group. Feed conversion (feed consumed/gain) was less efficient for inoculated turkeys than for ad libitum or pair-fed controls and was least efficient for turkeys inoculated with 5 X 10(5) oocysts. Plasma glutamic oxaloacetic transaminase (GOT, aspartate and aminotransferase) activity increased, and carotenoid and total protein levels decreased in inoculated turkeys but not in the pair-fed turkeys, indicating that these changes were caused by the infection and not by reduced food intake. Plasma glutamic pyruvic transaminase (GPT, alanine aminotransferase) remained stable in all groups. Plasma glucose levels of inoculated birds did not differ from those of the control groups, but liver glucose and glycogen levels decreased in both the inoculated and pair-fed birds.

The effects of thyroid hormones on prolactin (PRL) and growth hormone (GH) synthesis by the rat anterior pituitary gland were assessed in vitro. A marked reduction (84-87%) in the rate of H3-leucine incorporation into GH was evident 2-4 weeks after thyroidectomy, while incorporation into PRL was 52-71% less than that measured in glands from intact rats. A single injection of T4 (200 mug/kg) administered to thyroidectomized (THX) rats 48 hr before sacrifice significantly increased incorporation into both pituitary hormones, although the stimulation of GH synthesis was much more dramatic. Perphenazine, alpha-methyltyrosine and estrogen enhanced the rate of PRL synthesis in intact rats. Thyroid ablation did not affect the response to perphenazine, but significantly increased the response to alpha-methyltyrosine and estrogen. On the other hand, administration of T4 to THX rats receiving perphenazine, alpha-methyltyrosine or estrogen diminished the stimulatory influence of these treatments on PRL synthesis. Perphenazine, alpha-methyltyrosine and estrogen had no effect on the rate of GH synthesis in THX rats, nor did they alter the ability of T4 to restore GH synthesis in these animals. These results indicate that GH synthesis in the rat is dependent upon thyroid hormones and support the concept that these hormones exert their stimulatory effect directly on pituitary somatotrophs. Pituitary lactotrophs, however, appear to retain much of their capacity to synthesize PRL under conditions of thyroid deficiency. The changes in pituitary PRL levels and synthesis rate induced by thyroid ablation might reflect differences in the number rather than the activity of these cells.

The species-specificity and crossreactivity of serum antibodies (Ab) from birds immunized specifically with six different species of coccidia and of 24 hybridoma antibodies (Ab) developed against four species of chicken and two species of turkey coccidia were determined by use of the indirect immunofluorescent antibody (IFA) test on air-dried sporozoites. With few exceptions, the immune chicken sera were found to crossreact with all species of coccidia tested. Seven of the hybridoma Ab were species-specific, while the other 17 Ab demonstrated varying degrees of crossreactivity. Similar types of IFA patterns were seen with both the species-specific and crossreactive hybridoma Ab. Some of the crossreactive hybridoma Ab produced one type of IFA pattern with the sporozoites against which they were originally raised and different patterns with other species of sporozoites. The development of the hybridoma Ab has made it possible to identify the species of coccidia found in mixed infections and check the purity of laboratory strains.

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.

Since the original discovery of growth hormone (GH) by Drs. Herbert Evans and Joseph Long in 1921, this molecule has been a subject of keen interest to biochemical endocrinologists. There are a number of reasons for this interest. To state but a few: (1) the molecule does not have a single, well-defined target organ, and its biological and metabolic effects are quite numerous (e.g., regulation of growth as well as metabolism of protein, carbohydrate, and fat); (2) given the availability of several assay systems, the molecule is well suited to structure-function studies; (3) the molecule has an established clinical usefulness for the treatment of hypopituitary children in addition to its suggested use in the treatment of ulcers, muscular dystrophy, and coronary-prone hypercholesterolemic patients (Li, 1975); and (4) the cell that produces GH, viz., the pituitary somatotroph, offers an interesting model for investigation of mechanisms of intracellular processing and secretion of peptide hormones in addition to the study of the regulation of somatotroph function. This chapter deals with the last issue.

Hybridoma antibodies (Hab) produced against sporozoites or merozoites of four species of Eimeria were tested for the ability to inhibit the invasion of cultured primary avian kidney cells by sporozoites of Eimeria. Five of 16 Hab that were tested showed inhibitory activity. All five of these Hab were produced against sporozoites and reacted with sporozoite surface antigens or surface/internal antigens. Four Hab produced against merozoites of E. acervulina cross-reacted with sporozoite surface antigens but failed to inhibit invasion. Similarly, Hab reacting with sporozoite anterior tips or refractile bodies had little effect on invasion. Collectively, the data suggest that surface antigens or surface/internal antigens that are unique to the sporozoite stage may influence or be part of the invasion process. Indirect immunofluorescent-antibody tests and ferritin (Fe) labeling combined with electron microscopy indicated differences in binding of two of the Hab to the sporozoite surface membranes. For example, after exposure to Hab 43A6 and a fluorescein-antimouse IgG conjugate, extracellular sporozoites of E. meleagrimitis fluoresced brightly but intracellular sporozoites exhibited little fluorescent label. Sporozoites labeled with Hab 43A6 plus a ferritin-antimouse IgG conjugate that were observed in the process of cell invasion had ferritin on the extracellular portion of the parasite but not on the intracellular portion. Extracellular aggregates of ferritin were observed near the site of invasion. The data suggested that antigens of the sporozoite surface that are recognized by Hab 43A6 are "scraped off" during the invasion of cells. In contrast, after exposure to Hab E5, both extracellular and intracellular sporozoites of E. tenella fluoresced. However, ferritin label was not observed on viable sporozoites, even when they were fixed immediately after the labeling procedure. The antigens recognized by Hab E5 may be associated with parasite secretory products rather than with an integral part of the sporozoite surface membrane.

The production of PRL and GH by rat pituitary cell cultures was evaluated over a 9-day period in 20 commercially available media supplemented with 5% horse serum (HS) or 5% calf serum. The quantity of PRL produced by cells maintained in a-modified Eagle medium (α-MEM) containing 5% HS was consistently greater than that in all other combinations of media and sera examined. PRL production in α-MEM was increased from 4.7 to 8.1 μg PRL/25,000 pituitary cells during a 9-day culture (72% ↑) as HS concentrations were increased from 1.25% to 20% however, the rate at which PRL was released into the medium during culture was unaffected by HS concentrations above 2.5%. Maximum hormone production was achieved when pituitary cells were cultured at 37 C in CO2-NaHCO3-buffered α-MEM at a pH of 7.8. Changes in medium osmolality from 250 to 350 mosm/kg H2O did not significantly affect PRL secretion. Under the conditions employed in this study, rat somatotrophs did not significantly increase their original GH content durin...

The osmotic fragility of red blood cells (RBC) from Eimeria adenoeides-infected turkey poults was compared with that of RBC from control and water-deprived poults. At different hypotonic NaCl concentrations, lysis of RBC from infected poults was 10 to 35% less on day 4 postinoculation (PI) and 50 to 65% less on day 7 PI than that of controls. Red blood cells of poults deprived of water for 3 days were also resistant to lysis; the percent lysis was roughly the same as that of RBC from infected poults at day 7 PI. Incubating control RBC in plasma from infected poults, in extracts of infected ceca, or at different pH levels did not increase their resistance to lysis, suggesting that neither a stabilizing factor in the plasma that had rapid effect on the RBC nor a transient shift in blood pH was involved. Mean RBC size differed little among infected, water-deprived, and control poults (14.0-14.2 X 8.0-8.1 X 3.8 microns). However, although 3.5% of RBC population of control and water-deprived poults were immature (mid to late polychromatic erythrocytes), only 0.4% of the RBC of infected poults were immature. The data suggest that reduced water intake as well as other factors may be involved in the decreased osmotic fragility of RBC from poults infected with E. adenoeides.

This paper concerns itself with the personal life and professional contributions of one of the great men in optometry, R. C. Augustine. This short biography includes discussions on his childhood, his introduction to the field of optometry, his early contributions in his community, his book, his achievements during the presidency of the American Optometric Association and also his extension work the following two years. In summation, a discussion of his professional philosophy will follow.

Development of Eimeria tenella was studied in primary cultures of chicken kidney cells maintained in Medium 199 lacking each of the following: vitamin A, biotin, p-aminobenzoic acid, folic acid, nicotinamide, Ca pantothenate, pyridoxine, pyridoxal, riboflavin, thiamin, ascorbic acid, calciferol, alpha-tocopherol, and menadione. Data obtained concerning numbers of mature schizonts or total numbers of parasites or both indicated that all of the vitamins are needed for 1st- and 2nd-generation schizogony, and all except calciferol and folic acid are needed for gametogony.

Eimeria dispersa (turkey strain) and Eimeria gallopavonis sporozoites were inoculated into primary cultures of chicken kidney (CK) and turkey kidney (TK) cells. Eimeria dispersa sporozoites were more infective in either cell type than those of E. gallopavonis: at 4 hr, the percentage of infection was 67-98 for E. dispersa but only 23-56 for E. gallopavonis. E. dispersa also survived better in culture: at 2 days, losses of E. dispersa in both cell types were only 4-19%, whereas losses of E. gallopavonis were 35-47% IN TK cells and 60-95% CK cells. However, E. gallopavonis developed further than E. dispersa. Location and increase in numbers of intracellular stages at 4 days indicated that E. dispersa proceeded through 2 schizogonic generations before development stopped.

Electrophoretic separation of serum proteins of Eimeria-infected turkeys showed five major fractions with significant decreases in albumin levels and significant increases in the alpha 2- and gamma-globulins as compared with controls. The alpha 1- and beta-globulin levels were similar to those of the controls. Significant differences between infected turkeys and uninfected pair-fed controls suggested that protein changes were not solely due to the anorexia associated with Eimeria infections. The increase in alpha 2-globulins was not due to an increase in ceruloplasmin. Indirect fluorescent antibody tests using serum from infected poults as an antibody source were positive, indicating that at least part of the increase in the gamma-globulin fraction was due to the production of immunoglobulins. Electrophoretic separation of plasma lipoproteins showed three to four major bands with a sharp decrease in the portomicrons among infected poults. There was no increase in the pre-beta-lipoproteins (which have the same electrophoretic mobility as the alpha 2-globulins), indicating that the increase in alpha 2-globulins was not due to an increase in the lipoprotein part of this fraction.

The sterility of trypticase soy broth (TSB) that was frozen and thawed in disposable plastic syringes and infused via syringe pump was studied to determine whether ambient air or personnel-transferred contaminants compromised the sterility of the solution. Samples of TSB (10, 20, 30 mL) were prepared aseptically in syringes of three different brands--150 samples for each volume (50 for each manufacturer). The syringes were placed in zip-lock bags, stored for 24 hours at -15 to -20 degrees C, and thawed for three hours. Both positive and negative controls were used. For the test samples, infusion sets were connected to the syringes under aseptic conditions, and the solution was infused via syringe pump in ambient air into polyvinyl chloride minibags before incubation. The remaining samples were prepared in the same manner as the test solutions except that they were intentionally challenged with Bacillus subtilis introduced distal to the plunger. All samples were inspected visually for turbidity after a 14-day incubation period. There was no growth in any of the test infusion samples or in samples that were intentionally contaminated. The negative controls showed no growth; all of the positive controls showed growth. The sterility of solutions frozen in disposable plastic syringes does not appear to be compromised by touch contamination of the plunger shaft or by airborne microorganisms settling on the infusion system.

Developmental stages of Eimeria meleagrimitis Tyzzer were found throughout the intestine and ceca of turkeys given inocula ranging from 10(4) to 7.5 x 10(5) sporulated oocysts/bird. Infection initially occurred in the duodenum and upper jejunum but later moved down the intestine and into the ceca. The speed with which the infection moved into these areas was rougly proportional to the inoculum size. Heaviest infections were in the ileum, neck of the cecum, and large intestine. The life cycle consisted of 5 asexual generations before gametogony, a 6th asexual generation developing simultaneously with gametogony. First- and 2nd-generations were located along the sides of villi in the upper intestine rather than in the crypts of Lieberkühn, as previously described in England for this species. Transitory first-generation stages that were abnormally large and usually degenerate were found in the neck of the cecum.

Physiologic characteristics were measured on day 7 postinoculation (PI) in 2-week-old turkeys inoculated with 10(5) Eimeria adenoeides oocysts, in pair-fed controls, and in control turkeys fed ad libitum. Pathophysiologic responses were measured in turkeys inoculated with 7 x 10(4) E. adenoeides oocysts or 1.2 x 10(5) E. meleagrimitis oocysts and necropsied 2, 4, 6, 8, 10, 15, and 21 days PI. At day 7 PI, weight gains and heart weights (as a percentage of body weight) of E. adenoeides-infected turkeys were significantly lower and plasma glucose levels significantly higher than those of pair-fed counterparts. Plasma carotenoid and protein levels of the infected turkeys were significantly lower than those of the controls fed ad libitum and were consistently, but not always significantly, lower that those of the pair-fed controls. Significant responses were first observed in E. meleagrimitis-infected turkeys on day 4 PI and in E. adenoeides-infected birds on day 6 PI. Birds infected with both species commonly had reduced weight gains, heart weights, and plasma carotenoid levels and increased plasma aspartate aminotransferase levels. Plasma carotenoids in E. adenoeides-infected turkeys were significantly reduced on day 6 PI only, whereas plasma carotenoids in E. meleagrimitis-infected turkeys were sometimes reduced significantly as early as day 2 PI and remained significantly lower than control values through day 21 PI.

A method for the sterilizing filtration of Renacidin, a urologic irrigating solution, was evaluated. Renacidin irrigation was prepared and sterilized by microporous membrane filtration. A sterilizing membrane filtration apparatus was challenged by inoculating a batch of irrigation solution with Escherichia coli. The sterility of both intentionally contaminated and routinely prepared batches was evaluated. The stability of the solution was monitored by pH measurement, visual examination, maintenance of a vacuum, and absorbance spectrum of a 1:100 dilution in deionized water over a wavelength range from 400 to 200 nm. The time required to prepare three one-liter units was about two hours. No microbial growth was detected in any of the samples. The predicted minimum shelf-life at 10 degrees C was six months. Because the prepared solution contains some unreacted citric acid and bicarbonates, storage at room temperature could produce excessive pressure inside the container from carbon dioxide gas evolution. Refrigerated storage is recommended. This method for the preparation and sterilization of Renacidin irrigation is reasonably expedient, economical, and reliable.

White leghorn hens were experimentally infested with northern fowl mites (Ornithonyssus sylviarum) and antibody responses to mite immunogens were monitored over 12 weeks. Mite burdens increased during the early phase of infestation and declined over the latter weeks of the study. Antigen was prepared from homogenized whole mites, which were then sonicated and extracted with non-ionic detergent. Antigen extract was fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and antibody-reactive polypeptides were identified by immunoblotting. At the start of infestation, hens had natural, pre-existing antibodies that reacted with several mite-extract components. Individual hens had different natural antibody reactivities; however, all birds had immunoglobulins reactive with extract polypeptides of 117,000, 77,000 and 36,000 molecular weight. A variety of mite extract components reacted with hen antibodies generated in response to experimental infestation. The number of antibody-reactive mite polypeptides increased through week 8 of infestation and then decreased by week 12. Fifteen polypeptides of northern fowl mite extract were reactive with antibodies developed by the majority of infested birds. These commonly reactive polypeptides had molecular weights ranging from 40,000 to 160,000. Glycoconjugates of fractionated mite extract were identified by blotting with lectins that have different carbohydrate binding specificities. Also identified were lectins that bound extract components with the same molecular weights as those moieties complexed by immunoglobulins of infested birds.

The use of hybridoma antibodies developed against the sporozoite stage of avian coccidia, coupled with genetic-engineering techniques, has made it possible to begin bird-immunization studies utilizing an Escherichia coli-elicited coccidial protein. The coccidia are currently controlled in the poultry industry by use of anticoccidial compounds, but it now may be possible to use the bird's own immune system for defense against the parasitic infection. Since the sporozoite stage, which initiates the infection in poultry, is quite complex and is made up of hundreds of proteins or antigens, hybridoma antibodies were produced to identify specific antigens. These antigens, once identified, were found in such minute amounts that it became necessary to utilize genetic engineering in order to produce enough protein for immunization studies. One such protein, designated 5401, has been shown to stimulate an antibody response in immunized birds and to impart partial protection against a coccidial challenge infection. The results of these studies indicate that development of a vaccine against coccidial parasites may someday be possible.

The effect of acetaminophen on the labeling efficiency of leukocytes with indium oxine In 111 was studied. A blood sample was obtained from eight healthy men before and after they received acetaminophen 650 mg every four hours for 24 hours. After dividing the plasma from each sample into three portions, leukocytes were separated and labeled with indium oxine In 111. In an in vitro study, 200 ml of blood was obtained from one of the men, and the plasma was separated into four portions. Acetaminophen in 95% ethanol was added to three of the plasma fractions to produce acetaminophen concentrations of 4, 20, and 100 micrograms/ml; ethanol was added to the fourth fraction as a control. Each plasma fraction was then subdivided into three aliquots, and leukocytes were labeled as in the in vivo study. Mean leukocyte labeling efficiencies in both studies were calculated from the ratios of leukocyte radioactivity to initial radioactivity in the samples, expressed as percentages. Leukocyte labeling efficiencies before acetaminophen administration ranged from 79 to 85%; after administration, labeling efficiencies ranged from 70 to 87%. No significant differences in mean labeling efficiency before and after acetaminophen administration were noted in any of the subjects. Leukocyte labeling efficiencies in all in vitro plasma fractions were reduced, ranging from 54 to 63%, but no significant differences in labeling efficiency between any of the plasma fractions were found. Using the labeling procedures in this study, exposure of leukocytes from healthy men to acetaminophen in vivo or in vitro does not affect labeling efficiency with indium oxine In 111.

Beltsville small white and Nicholas turkey poults, 1, 7, and 14 days old, were inoculated with mixed cultures of Eimeria adenoeides and E. meleagrimitis. Weight gain and feed conversion of 1-day-old poults were affected as severely as those of the older poults, and mortality was heaviest in this group, ranging from 25 to 45%. One dose of oocysts (2 x 10(4)-1.5 x 10(5], given at 1, 7, or 14 days of age, protected even the 1-day-old poults against challenge with 3 x 10(5) oocysts. Protection was comparable to that afforded by multiple immunizing doses given over 3 weeks. After challenge, little weight reduction or mortality was observed in immunized poults. Average feed conversions of the immunized challenged poults were 1.52 to 1.69 as compared with 2.98 to 5.14 for unimmunized challenged poults.

Two-wk-old broiler chicks were inoculated via crop intubation with Eimeria acervulina at two doses: 10(5) or 10(6) sporulated oocysts/bird or with Eimeria tenella at a dose of 10(5) sporulated oocysts/bird. Serum and liver samples were collected on days 3 and 6 post-inoculation (PI). There were no significant changes in serum or liver zinc, copper, and iron concentrations in any of the infected groups by 3 d PI. However, on d 6, PI serum protein was significantly reduced in all of the infected groups compared to their pair-fed controls. The chicks infected with E. tenella had significantly reduced serum zinc (1.20 vs 1.77 micrograms/mL) and iron (0.44 vs 1.28 micrograms/mL) concentrations and significantly elevated serum copper (0.28 vs 0.17 micrograms/mL) and ceruloplasmin levels (20.33 vs 11.11 micrograms/mL) compared to their pair-fed counterparts. Those chicks infected with E. acervulina (10(6) oocysts/bird) exhibited significantly reduced serum iron concentration by 6 days PI (0.90 vs 1.14 micrograms/mL). Liver zinc was significantly increased in the chicks infected with E. tenella (349 vs 113 micrograms/g dry liver wt), as was copper (24 vs 19 micrograms/g), whereas liver iron concentration was significantly reduced (172 vs 243 micrograms/g) compared to pair-fed controls. At both dose levels, the chicks infected with E. acervulina exhibited a significant reduction in liver iron by 6 d PI. Hepatic cytosol metals generally reflected whole tissue levels. Metallothionein (MT)-bound zinc was significantly elevated in the chicks infected with E. tenella. Iron bound to a high molecular weight, heat-stable protein fraction (presumably cytoplasmic ferritin) was significantly reduced in chicks infected with E. acervulina, as well as those infected with E. tenella. Collectively, the changes in serum zinc, copper, and iron concentrations, as well as the changes in hepatic zinc and MT-zinc concentrations in the chicks infected with E. tenella were similar to changes evoked during an acute phase response to infection. It is possible that a secondary bacterial infection or inflammation stemming from erosion of the lining of the cecum may play a role in the response of trace element metabolism to the E. tenella infection.

Glucose and methionine were malabsorbed in some intestinal regions of turkeys infected with Eimeria meleagrimitis, E. adenoeides , or E. dispersa . The decrease in absorption was not always related to the numbers of parasites in the cells or the extent of damage to the mucosa. With E. adenoeides , malabsorption was found in the jejunum even though parasites were not present. Conversely, with E. dispersa , no malabsorption was observed in the duodenum even though light microscopy showed numerous parasites. In many intestinal regions, damage to the mucosal surface visible with scanning electron microscopy (SEM) was slight or absent, although malabsorption was marked. No changes were noted with SEM in the structure and orientation of the brush border in these regions. Villar height was significantly reduced in the regions of heaviest infection when intestinal damage was visible. Conversely, the crypts of Lieberkuhn were often two or three times as deep in infected poults as in uninfected poults. In general, no differences were found in the thickness of the circular and longitudinal muscle layers between the infected and uninfected poults. The dry weight of the intestinal tissue was less from infected poults than from uninoculated controls and was related to both region of the intestine and severity of the infection.

The electrophoretic distribution of serum proteins was measured in chickens inoculated with sporulated oocysts of Eimeria acervulina, E. tenella, and E. maxima. Total serum protein decreased on day 5 and 7 postinoculation (PI) with all three species. However, the changes produced in the individual serum protein fractions differed with the species. In serum from E. acervulina-infected birds, all protein components decreased quantitatively; the largest decrease was in the alpha globulins. The overall percent distribution of the globulins was similar to that of uninoculated controls, except for a decrease in the alpha globulins. In E. tenella-infected chickens, albumin decreased (both quantitatively and in percent distribution), but the alpha 1, beta, and gamma 1 globulins increased. In E. maxima-infected chickens, only albumin and alpha 1 globulin decreased quantitatively; the amounts of alpha 2, beta, or gamma globulins did not change. These changes were transitory and had disappeared by day 10 PI.

Heart composition and function were measured in turkey poults inoculated with Eimeria meleagrimitis or E. adenoeides oocysts. Heart weights of infected poults were significantly lower than those of uninfected controls. Lipid levels of the heart were significantly reduced on day 4 postinoculation (PI) with E. meleagrimitis and on day 6 PI with E. adenoeides. The reduced lipid levels were closely associated with decreases in body weight. The protein per gram of heart tissue increased as the lipid levels decreased. Heart glycogen, when expressed as microgram per milligram of protein, decreased. However, the concentration of glycogen per gram of tissue apparently changed very little. Moisture levels of hearts from infected and control poults did not differ significantly. Heart rates and mean blood pressures of poults infected with E. adenoeides were slightly (approximately 8%) lower than values in control poults. However, exposure to stress in the form of ice baths or intravenous injection of epinephrine caused more severe and prolonged fluctuations in heart rate and blood pressure of the infected poults than in those of the controls.

SUMMARYEimeria meleagrimitis oocysts were stored at 4, 22, 32 or 41·5 °C for up to 1 year. Decreases in amylopectin levels (measured as glucose) and viability (measured as oocyst production and mortality in turkeys) of the oocysts were generally related to the length of time in storage and the storage temperature. Oocysts assayed immediately after harvest contained 58·29 ± 0·75 μg of glucose/106 oocysts. When the oocysts were stored at 4 °C for 162 days, the amylopectin level decreased to 65% of the original level. In oocysts stored at 22, 32 and 41·5 °C, amylopectin declined to approximately 20% within 162, 76, and 41 days, respectively. Oocysts stored at 4 °C for 1 year produced more oocysts in turkeys than the original fresh isolate, but caused no mortality. Oocyst production from oocysts stored at 22 and 32°C decreased gradually until, after 9 and 7 months respectively, no patent infections were produced. Oocyst production from oocysts stored at 41·5 °C was markedly reduced within 1 month and was not detected after 4 months.

An irreversible beta-adrenergic blocker, bromoacetylalprenololmenthane (BAAM), was administered both peripherally and centrally to turkey poults, Meleagris gallopavo. Peripheral administration of BAAM (60 mg/kg body weight) effected a significant reduction in blood pressure and heart rate. Twenty minutes postinjection, mean blood pressure and heart rate were reduced 34.5 and 24.2%, respectively. Two days later, mean blood pressure values remained significantly depressed at 17.3% below preinjection determinations. Biochemical analysis of heart tissue following peripheral (intraperitoneal) injections of BAAM (60 mg/kg body weight) showed a significant decrease in beta-adrenergic receptors (BAR). Little or no change in the number of BAR in brain tissue was observed. Central (intraventricular) administration of BAAM (0.72 mg/g brain weight) resulted in no change in mean blood pressure or heart rate during a 20 min postinjection period. Biochemical analysis of heart tissue following central injections of BAAM showed little or no change in the number of BAR. There was, however, a significant decrease in the number of BAR in brain tissue.

The invasion of the intestinal epithelium of immunized and unimmunized turkeys and chickens by four species of Eimeria was quantitated. In unimmunized birds, E. adenoeides, E. acervulina, and E. tenella invaded primarily the areas in which first-generation schizonts subsequently developed. Eimeria meleagrimitis invaded a larger area of the intestine. Between 1 and 4 hr postinoculation, the numbers of intracellular sporozoites increased, but their location within the intestine was little changed. When birds were immunized with either of two lower intestinal species, E. adenoeides or E. tenella, and then challenged with the immunizing species, invasion was reduced by 36% to 55%. In contrast, immunizing and then challenging birds with either of two upper intestinal species, E. meleagrimitis or E. acervulina, did not reduce invasion: there were 44% more intracellular sporozoites in E. meleagrimitis-immunized turkeys and 11% more in E. acervulina-immunized chickens than in their unimmunized counterparts.

Monoclonal antibodies were developed against refractile body antigens of 4 species of avian Eimeria, E. meleagrimitis, E. adenoeides, E. acervulina, and E. tenella. Although antibodies from 8 different cell lines were used in this study, all produced similar fluorescent and gold-labeling patterns. By immunofluorescent antibody techniques, 5 of the 8 antibodies cross-reacted with all 4 of the Eimeria species that were examined; the other 3 antibodies reacted only with the species against which they were produced or with a limited number of species. In Western blot analyses using SDS-solubilized sporozoites as antigen, 4 of the cross-reactive antibodies recognized multiple bands; the predominant bands had molecular weights of approximately 23, 45, and 90 kilodaltons (kDa). Two of the antibodies with more limited reactivity recognized either a single band at 23 kDa (91C7), or bands at 23 and 45 kDa (4115); another reacted only with several bands greater than 100 kDa (4D10). The molecular weights of the antigens did not decrease markedly after digestion with N-glycanase F, indicating that if the refractile body antigens contained significant amounts of N-linked carbohydrate it was refractory to the enzyme. Collectively, the data indicate that antigens of the sporozoite refractile bodies differ among the Eimeria species. Some antigens are conserved, whereas others differ in distribution or frequency among the individual species.

SYNOPSIS. During a 7-year period Histomonas meleagridis survived 1000 passages in Medium 199 fortified with serum and antibiotic-killed bacteria. The histomonads were originally isolated from a chicken's cecal dropping, and the bacteria were cultivated from the cecal contents of a normal turkey. In many respects, the histomonads remained unchanged during cultivation, but in some other respects they changed considerably, perhaps irreversibly. Morphologically, the histomonads propagated in vitro showed only slight changes. When returned to birds, they resumed the structure characteristic of lumen-dwelling individuals of this species. However, they had long since lost their ability to produce disease in either chickens or turkeys, and organisms of the tissue-dwelling type were rarely seen. The long-cultivated histomonads are actually as nonpathogenic as Histomonas wenrichi, but they have none of the distinguishing morphologic characteristics of the latter. As has been reported elsewhere, H. meleagridis long maintained in the tissue culture medium has also lost much of its ability to immunize either chickens or turkeys against infection with virulent strains of this parasite. Also lost in the process of adaptation to its restricted medium has been the ability to multiply satisfactorily in vitro with the complement of bacteria normal to the ceca of the birds. However, in some instances, histomonads which have become adapted to in vitro cultivation are still able to live in birds with this diversified flora. Activity of the histomonads cultivated in vitro differed but little from that of H. meleagridis freshly isolated from birds, when both were viewed under the same conditions. Histomonads from each of the above sources multiplied most satisfactorily in their accustomed habitat, probably because of a difference in nutritional requirements.

SYNOPSIS Eimeria tenella completed its endogenous life cycle in primary cultures of kidney cells from 2- to 3-week-old-chickens, guinea fowl, partridges, pheasants, quail, and turkeys. Similarity in percentage of infection at 4 hr suggested that sporozoites entered cells from all birds in equal numbers. Development was better, however, in chicken cells in that the percentage of survival and of developmental stages during the first 2 days were greater, developmental stages occurring after 2 days usually were found earlier, mature 2nd-generation schizonts and oocysts were larger, and oocyst production was far greater than in nonhost cells. Multinucleate macrogametes, which sometimes reached sizes 3–4 times greater than normal oocysts, are reported for the first time.

Histomonas meleagridis was cultivated more than 6 years in Tissue Culture Medium 199, fortified with serum and antibiotic-killed bacteria of cecal origin. The histomonads first lost their ability to produce disease but retained some immunizing ability. During the sixth year of in vitro cultivation, representing serial passages 730–835, the immunizing ability declined. At the same time, these attenuated histomonads were also losing their ability to survive in the presence of freshly isolated, antibiotic-killed cecal bacteria. Meantime, freshly isolated histomonads were unable to live in the culture medium with only the limited flora that had survived the numerous in vitro passages. Apparently, long continued cultivation operated selectively on both the bacteria and Histomonas, leaving only histomonads with no ability to penetrate the host's tissues.

SYNOPSIS. Histomonas meleagridis cultured in vitro for 8 years, H. meleagridis freshly isolated from cecal contents of turkeys, and freshly isolated, nonpathogenic H. wenrichi were compared by indirect fluorescent antibody tests. The Histomonas antisera were produced in rabbits in response to cultured and freshly isolated H. meleagridis. Using fluorescein-conjugated antirabbit globulin, fluorescent reactions of cultured and freshly isolated H. meleagridis with either H. meleagridis antisera were indistinguishable. Such results suggest that the antigens involved in these reactions remained relatively unchanged during prolonged cultivation. H. wenrichi reacted feebly with both H. meleagridis antisera.

Monoclonal antibodies were developed against refractile body antigens of 4 species of avian Eimeria, E. meleagrimitis, E. adenoeides, E. acervulina, and E. tenella. Although antibodies from 8 different cell lines were used in this study, all produced similar fluorescent and gold-labeling patterns. By immunofluorescent antibody techniques, 5 of the 8 antibodies cross-reacted with all 4 of the Eimeria species that were examined; the other 3 antibodies reacted only with the species against which they were produced or with a limited number of species. In Western blot analyses using SDS-solubilized sporozoites as antigen, 4 of the cross-reactive antibodies recognized multiple bands; the predominant bands had molecular weights of approximately 23, 45, and 90 kilodaltons (kDa). Two of the antibodies with more limited reactivity recognized either a single band at 23 kDa (91C7), or bands at 23 and 45 kDa (4115); another reacted only with several bands >100 kDa (4D10). The molecular weights of the antigens did not decrease markedly after digestion with N-glycanase F, indicating that if the refractile body antigens contained significant amounts of N-linked carbohydrate it was refractory to the enzyme. Collectively, the data indicate that antigens of the sporozoite refractile bodies differ among the Eimeria species. Some antigens are conserved, whereas others differ in distribution or frequency among the individual species. Refractile bodies (RB) are the most conspic- uous structures in the sporozoites of most Ei- meria species, occupying approximately 30-50% of the cytoplasmic area. These structures have been described as clear globules (Hammond, 1973) that are proteinaceous and have no indi- cation of structure (Ryley, 1973). The function of the RB is unknown. In the species of avian Eimeria that have been studied, the RB appear to undergo changes in number, shape, and lo- cation during the first few days after invading a host cell (Fayer, 1969). Investigators have spec- ulated that the RB, because of the rapid changes after entering a cell, may play a role in the early development of the parasite (Chobotar and Scholtyseck, 1982). A recent study has indicated that refractile body antigens of E. tenella must diffuse throughout the first-generation schizont for maturation to occur. Collectively, these stud- ies suggest that inhibition of RB activity might result in decreased intracellular development of the parasite. The RB antigens of sporozoites of the avian Eimeria species have not been de- scribed. In the study reported here, RB antigens of 4 species of avian Eimeria, E. meleagrimitis, E. adenoeides, E. acervulina, and E. tenella, were examined. Monoclonal antibodies generated against the RB were used as probes for differences

SUMMARY Changes in levels of plasma xanthophyll, plasma retinol, liver retinol, and liver retinylpalmitate in poults infected with Eimeria meleagrimitis or E. adenoeides were measured by high-performance liquid chromatography. Infection with either species significantly reduced total plasma carotenoids; the reduction was due largely to a decrease in xanthophyll to approximately 50% of control levels on the day of maximum effect. In addition, E. meleagrimitis infection decreased plasma retinol levels from 1.84 ,g/ml plasma (controls) to 1.00 ,ug/ml plasma on day 6 postinoculation. Concomitantly, liver retinol levels fell from 0.23 ,ig/mg protein (controls) to 0.15 jg/mg protein, and liver retinylpalmitate levels fell from 0.74 /ug/mg protein (controls) to 0.44 jg/mg protein. In contrast, Eimeria adenoeides infection caused little change in either plasma or liver retinol levels.

The invasion of the intestinal epithelium of immunized and unimmunized turkeys and chickens by four species of Eimeria was quantitated. In unimmunized birds, E. adenoeides, E. acervulina, and E. tenella invaded primarily the areas in which first-generation schizonts subsequently developed. Eimeria meleagrimitis invaded a larger area of the intestine. Between 1 and 4 hr postinoculation, the numbers of intracellular sporozoites increased, but their location within the intestine was little changed. When birds were immunized with either of two lower intestinal species, E. adenoeides or E. tenella, and then challenged with the immunizing species, invasion was reduced by 36% to 55%. In contrast, immunizing and then challenging birds with either of two upper intestinal species, E. meleagrimitis or E. acervulina, did not reduce invasion: there were 44% more intracellular sporozoites in E. meleagrimitis-immunized turkeys and 11% more in E. acervulina-immunized chickens than in their unimmunized counterparts.

White leghorn hens were experimentally infested with northern fowl mites (Ornithonyssus sylviarum) and antibody responses to mite immunogens were monitored over 12 weeks. Mite burdens increased during the early phase of infestation and declined over the latter weeks of the study. Antigen was prepared from homogenized whole mites, which were then sonicated and extracted with non-ionic detergent. Antigen extract was fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and antibody-reactive polypeptides were identified by immunoblotting. At the start of infestation, hens had natural, pre-existing antibodies that reacted with several mite-extract components. Individual hens had different natural antibody reactivities; however, all birds had immunoglobulins reactive with extract polypeptides of 117,000, 77,000 and 36,000 molecular weight. A variety of mite extract components reacted with hen antibodies generated in response to experimental infestation. The number of antibody-reactive mite polypeptides increased through week 8 of infestation and then decreased by week 12. Fifteen polypeptides of northern fowl mite extract were reactive with antibodies developed by the majority of infested birds. These commonly reactive polypeptides had molecular weights ranging from 40,000 to 160,000. Glycoconjugates of fractionated mite extract were identified by blotting with lectins that have different carbohydrate binding specificities. Also identified were lectins that bound extract components with the same molecular weights as those moieties complexed by immunoglobulins of infested birds.

SUMMARY The osmotic fragility of red blood cells (RBC) from Eimeria adenoeides-infected turkey poults was cor ipared with that of RBC from control and water-deprived poults. At different hypotonic NaCI concentrations, lysis of RBC from infected poults was 10 to 35% less on day 4 postinoculation (PI) and 50 to 65% less on day 7 PI than that of controls. Red blood cells of poults deprived of water for 3 days were also resistant to lysis; the percent lysis was roughly the same as that of RBC from infected poults at day 7 PI. Incubating control RBC in plasma from infected poults, in extracts of infected' ceca, or at different pH levels did not increase their resistance to lysis, suggesting that neither a stabilizing factor in the plasma that had rapid effect on the RBC nor a transient shift in blood pH was involved. Mean RBC size differed little among infected, water-deprived, and control poults (14.0-14.2 X 8.0-8.1 x 3.8 im). However, although 3.5% of RBC population of control and water-deprived poults were immature (mid to late polychromatic erythrocytes), only 0.4% of the RBC of infected poults were immature. The data suggest that reduced water intake as well as other factors may be involved in the decreased osmotic fragility of RBC from poults infected with E. adenoeides. RESUMEN

Beltsville small white and Nicholas turkey poults, 1, 7, and 14 days old, were inoculated with mixed cultures of Eimeria adenoeides and E. meleagrimitis. Weight gain and feed conversion of 1-day-old poults were affected as severely as those of the older poults, and mortality was heaviest in this group, ranging from 25 to 45%. One dose of oocysts (2 x 10(4)-1.5 x 10(5], given at 1, 7, or 14 days of age, protected even the 1-day-old poults against challenge with 3 x 10(5) oocysts. Protection was comparable to that afforded by multiple immunizing doses given over 3 weeks. After challenge, little weight reduction or mortality was observed in immunized poults. Average feed conversions of the immunized challenged poults were 1.52 to 1.69 as compared with 2.98 to 5.14 for unimmunized challenged poults.

Cell cultures from kidneys of turkeys were treated with four cations, two polyanions, or two proteolytic enzymes and then inoculated with Eimeria meleagrimitis sporozoites. After a 20-min incu- bation, cultures treated with three of the cations (poly-L-histidine, poly-L-lysine, or Ca++) or with chy- motrypsin contained significantly fewer intracellular sporozoites than did the untreated control cultures. Increases in the concentration of poly-L-histidine or chymotrypsin produced linear decreases in the num- bers of sporozoites. In contrast, after a 2-hr incubation, treated and control cultures contained the same numbers of intracellular sporozoites. Treatment of cell cultures with the fourth cation (DEAE-dextran), the two polyanions (heparin or dextran sulfate), or trypsin did not significantly affect the numbers of sporozoites after either 20-min or 2-hr incubation. Sporozoites that were pretreated with poly-L-histidine, Ca++, or chymotrypsin for 20 min and then inoculated into untreated cultures were found intracellularly in the same numbers as were untreated sporozoites. Apparently, the reduced number of intracellular sporozoites in treated cultures resulted from interactions between the treatment substances and the host cells, and was not caused by immobilization of the parasite. The effect of various agents on entry of Ei- meria sporozoites into cells has been studied by several investigators. Jensen and Edgar (1976) treated cells with agents that reduce phagocytosis, and found that penetration of the cells by Eimeria magna sporozoites was not inhibited. Fayer et al. (1970), working with Eimeria adenoeides, reported that hyal- uronidase, hyaluronic acid, and chondroitin sulfate did not alter the number of intracel- lular sporozoites when these reagents were included in the inoculation medium, and the infected cells fixed 1 hr later. However, Fayer (1971) found that quinine compounds inhib- ited penetration of cells, and suggested that