Patricia C. Augustine

Development of resistance to coccidiosis in the absence of merogonic development using X-lrradiated Eimeria acervulina oocysts

Sporulated oocysts of the protozoan Eimeria acervulina were subjected to 0, 10, 15, 20, or 30 krad of X-irradiation and inoculated into susceptible outbred chickens to determine if radioattenuated coccidia could induce protection against parasite challenge. Irradiation treatment had an appreciable dose-dependent effect on parasite development. Insignificant numbers of oocysts were produced by chickens inoculated with parasites that had been exposed to greater than 10 krad X-irradiation. Sporozoites exposed to 15 or 20 krad irradiation conferred significant protection against the appearance of intestinal lesions after parasite challenge. Sporozoites subjected to the highest dose level (30 krad) did not produce any significant level of protection. To investigate this phenomenon further and assess intracellular parasite development, susceptible outbred strains of chickens were administered either nonirradiated (0 krad) oocysts or oocysts that were exposed to an optimal dose (15 krad) or a high dose (30 krad) of X-irradiation. Immunofluorescence staining of tissue sections from each treatment group at various intervals after the initial administration of irradiated parasites indicated that sporozoites exposed to 15 krad irradiation were as capable of invading the host intestinal epithelium as nonirradiated sporozoites. However, at 48, 60, 72, and 96 hr, there was a marked reduction in merogonic development in groups receiving irradiated sporozoites compared to those inoculated with nonirradiated parasites. The latter parasites underwent profuse merogonic development; in contrast, irradiated parasites demonstrated little (15 krad) or no (30 krad) merogonic development. These results suggest that induction of a protective immune response occurs during a critical period early in intracellular development of E. acervulina.

Immunizing action of in vitro-attenuated Histomonas meleagridis in chickens and turkeys☆

Abstract 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 hosts tissues.

Protective Immunity against Coccidiosis Elicited by Radiation-Attenuated Eimeria maxima Sporozoites that are Incapable of Asexual Development

Eimeria maxima oocysts were exposed to various doses of gamma radiation that did not affect sporozoite invasion of intestinal epithelium but did prevent subsequent merogonic development therein. Although merogony and oocyst formation were inhibited, parasites exposed to 12 kRad radiation induced a level of immunity against E. maxima challenge equivalent to that induced by non-irradiated oocysts. Chickens immunized per os with 20 kRad-treated E. maxima oocysts were not protected against coccidial challenge. Immunization of chickens with a single low dose (five oocysts) of non-irradiated (0 kRad) or irradiated (12 kRad) E. maxima was effective in preventing weight depression after coccidial challenge. Immunofluorescence staining of intestinal tissue from chickens infected with irradiated (12 or 20 kRad) or non-irradiated (0 kRad) E. maxima oocysts with developmental stage-specific monoclonal antibodies showed that sporozoite invasion was similar in all groups. However, merogonic development was not observed at any time postinfection in chickens infected with irradiated oocysts, unlike the case with chickens infected with non-irradiated parasites. These results suggest that sporozoite-infected host cells are capable of eliciting complete protection against E. maxima challenge.

The effect of dietary betaine on intestinal and plasma levels of betaine in uninfected and coccidia-infected broiler chicks

Chicks fed betaine supplemented diets and infected with Eimeria acervulina and Eimeria maxima had markedly higher levels of betaine in the duodenum and mid-gut than unsupplemented, infected chicks. Uninfected chicks fed betaine exhibited almost twice the levels of betaine in the gut as infected chicks. Plasma betaine levels were lower in E. maxima-infected chicks than in E. acervulina-or Eimeria tenella-infected chicks. Betaine supplementation reversed the decrease in weight gain in E. maxima- infected chicks but had no effect on the decrease in weight gains in E acervulina- and E. tenella-infected chicks. Coccidia-infected birds on normal diets regularly exhibit increases in plasma NO2+NO3. This increase was abolished in E.tenella-infected birds on betaine supplement. Betaine feeding did not alter this effect in E. acervulina- and E. maxima-infected birds. Results indicate that betaine supplementation has a positive effect on gut betaine levels in birds infected with E. acervulina and E. maxima. In all treatment groups, infection lowered the levels of betaine.

Development of protective immunity against Eimeria tenella and E. acervulina in white leghorn chickens inoculated repeatedly with high doses of turkey coccidia

Repeated inoculation (immunization) of 2-week-old white leghorn chickens with 10(6) oocysts of the turkey coccidia Eimeria adenoeides or E. meleagrimitis partially protected chickens against moderate challenge with E. tenella or E. acervulina oocysts, but not with E. necatrix oocysts. After challenge, mean weight gains of the immunized chickens and the unchallenged controls did not differ significantly, but weight gains of unimmunized chickens were significantly lower. The mean feed-conversion ratio of the immunized challenged chickens was 3.14, as compared with 4.42 for unimmunized challenged control chickens. In general, immunization did not markedly reduce intestinal lesions. Repeated inoculation of chickens with the turkey coccidium E. gallopavonis failed to produce statistically significant protection against challenge with E. tenella, E. acervulina, or E. necatrix, as determined by weight gain, feed-conversion efficiency, and lesion scores. Antibody profiles of individual chickens did not correlate with protection.

Serum and liver zinc, copper, and iron in chicks infected withEimeria acervulina orEimeria tenella

Two-wk-old broiler chicks were inoculated via crop intubation withEimeria acervulina at two doses: 105 or 106 sporulated oocysts/bird or withEimeria tenella at a dose of 105 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 withE. tennella had significantly reduced serum zinc (1.20 vs 1.77 μg/mL) and iron (0.44 vs 1.28 μg/mL) concentrations and significantly elevated serum copper (0.28 vs 0.17 μg/mL) and ceruloplasmin levels (20.33 vs 11.11 μg/mL) compared to their pair-fed counterparts. Those chicks infected withE. acervulina (106 oocysts/bird) exhibited significantly reduced serum iron concentration by 6 days PI (0.90 vs 1.14 μg/mL). Liver zinc was significantly increased in the chicks infected withE. tenella (349 vs 113 μg/g dry liver wt), as was copper (24 vs 19 μg/g), whereas liver iron concentration was significantly reduced (172 vs 243 μg/g) compared to pair-fed controls. At both dose levels, the chicks infected withE. 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 withE. tenella. Iron bound to a high molecular weight, heat-stable protein fraction (presumably cytoplasmic ferritin) was significantly reduced in chicks infected withE. acervulina, as well as those infected withE. 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 withE. 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 theE. tenella infection.

Biotechnology for Solving Agricultural Problems

Gene maps are constructed by the synthesis of data obtained by different methods which include family analyses, somatic cell hybridization, direct mapping of DNA segments by Southern blot analysis and in situ hybridization to fixed metaphase chromosomes. Gene mapping has already contributed significantly to a better understanding of the mammalian genome, in particular the human genome but the gene maps of economically important domestic species are not well characterized. The application of somatic cell genetics and recombinant DNA methodologies now allows rapid progress to be made in the construction of detailed gene maps for domestic animals. Such gene maps will serve as tools for selection in applied breeding and for the analysis of polygenic traits.

Invasion of different cell types by sporozoites of Eimeria species and effects of monoclonal antibody 1209-C2 on invasion of cells by sporozoites of several apicomplexan parasites.

Abstract Sporozoites of avian Eimeria species differed markedly in their ability to invade cells in vitro. Invasion by E. tenella and E. adenoeides was significantly greater in baby hamster kidney (BHK) and chicken cecal cell (CC) cultures than in primary chicken (PCK) or turkey kidney (PTK) cell cultures. Moreover, invasion of BHK cell cultures by E. adenoeides was significantly greater than that of other Eimeria species, and invasion by E. acervulina sporozoites was significantly lower. Monoclonal antibody 1209-C2 (MAb 1209-C2) reacted by immunofluorescent labeling (IFA) with refractile bodies of sporozoites of 5 species of Eimeria and Caryospora bigenetica, but not with sporozoites of Toxoplasma gondii, Hammondia hammondi, or Cryptosporidium parvum, which have no refractile bodies. The MAb also cross-reacted with formalin-fixed BHK, CC, turkey cecal (TC) cells, and PTK. Pretreatment of BHK cells with MAb 1209-C2 significantly reduced invasion of the cells by sporozoites of E. tenella, E. acervulina, E. meleagrimitis, and C. bigenetica, but did not alter invasion by T. gondii, C. parvum, or H. hammondia. Apparently, reactivity of MAB1209-C2 with the sporozoites was required for inhibition of invasion despite the fact that the inhibition resulted from pre-treatment of the host cell. Conversely, although MAb 1209-C2 also reacted moderately with PTK and TC cells, pre-treatment of these cell cultures with the MAb did not inhibit invasion by either MAB 1209-C2-reactive or -nonreactive parasites. Collectively, the data indicated that refractile body antigens of sporozoites of Eimeria and Caryospora, which are recognized by MAb 1209-C2, may function in cellular invasion, but also suggest that cellular invasion is probably not mediated by interactions between the conserved epitopes in sporozoites and cultured host cells that are recognized by the MAb.

Eimeria meleagrimitis, E. adenoeides, and E. dispersa: Severity of infection and changes in the intestinal mucosa of the Turkey

Abstract 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.

Avian eimeria: effects of gamma irradiation on development of cross-species immunity in foreign and natural host birds

Repeated inoculation (immunization) of white leghorn chickens with oocysts of the turkey coccidium, Eimeria adenoeides, resulted in significantly improved weight gain and feed-conversion ratios (feed efficiency) after E. tenella challenge. However, the development E. tenella in the immunized chickens did not differ markedly from that in unimmunized chickens, and intestinal lesions remained severe (greater than 3.0). Apparently improved weight gain and feed efficiency can be maintained in the presence of extensive parasite development. The ability to elicit cross-protective immunity was abrogated when the E. adenoeides oocysts were exposed to 15 kRad of gamma irradiation before inoculation into the chickens. Sporozoites existing from irradiated oocysts of E. acervulina also failed to immunize the chickens against challenge with E. tenella but protected chickens almost completely against homologous challenge with E. acervulina. The results indicate that cross-species immunity is not elicited by all developmentally arrested sporozoites and that the ability to produce cross-species immunity can be destroyed by gamma irradiation of the immunizing species.