H. D. Danforth

Dietary modulation of avian coccidiosis

During the past several years, our laboratory has been investigating the anticoccidial activities of various natural products that have potential use as dietary supplements for coccidiosis control. Sources of fats containing high concentrations of n-3 fatty acids such as menhaden oil and flaxseed oil and flaxseed, when added to starter rations and fed to chicks from one day of age, effectively reduce lesions caused by the caecal parasite Eimeria tenella, but not lesions caused by Eimeria maxima. Our results are consistent with reports of effects of diets high in n-3 fatty acids on other protozoan parasites which suggest that the state of oxidative stress induced by these diets in the cells of both host and parasites is responsible for their parasitic actions. Artemisinin, a naturally occurring (Artemisia annua) endoperoxide and effective antimalarial significantly lowers lesions from E. tenella when given at low levels as a feed additive. The mechanism of its action is also considered to involve induction of oxidative stress. Diets supplemented with 8 p.p.m. gamma-tocopherol (abundant in flaxseeds) or with 1% of the spice tumeric, reduce mid-small intestinal lesion scores and improve weight gains during E. maxima infections. These compounds may exert their anticoccidial activity because they are effective antioxidants. Betaine, a choline analogue found in high concentrations in sugar beets, improves nutrient utilisation by animals under stress. When provided as a dietary supplement at a level of 0.15% it has enhanced the anticoccidial activity of the ionophore, salinomycin. Betaine may act as an osmoprotectant whereby it improves the integrity and function of the infected intestinal mucosa. In in vivo studies, betaine plus salinomycin significantly inhibit invasion of both E. tenella and E. acervulina. However, subsequent development of E. acervulina is inhibited more effectively with this combination treatment than development of E. tenella.

Use of live oocyst vaccines in the control of avian coccidiosis: experimental studies and field trials

Areas addressed in this study on the use of live oocyst vaccines to control coccidiosis include: the influence of immunocompetency of the strains and sex of the birds used; methods of delivery of vaccine; immunological variation between different strains of the same coccidial species; and the effects of combining vaccine with anticoccidial medication. The results show that vaccination with live oocysts elicited significant protection against coccidiosis, both with experimentally induced and naturally acquired coccidial infection, resulting in average bird weight gains and feed efficiency similar to that obtained with conventional anticoccidial medication.

Ultrastructural observations of host-cell invasion by sporozoites ofEimeria papillata in vivo

Scanning and transmission electron microscopy were used to study the invasion of mouse small-intestinal epithelium by sporozoites ofEimeria papillata. Some mice received oocysts by gavage and others received either sporocysts or sporozoites by direct injection into the small intestine. The highest concentration of invaded cells were found in ligated intestinal tissues studied at 5–45 min after the inoculation of sporozoites. Sporozoites actively invaded anterior end first, which resulted in extensive damage to the host cell. Such cells showed disrupted microvilli; protuberances of cytoplasm into the lumen, apparently the result of a disrupted plasma membrane; vacuolization of the cytoplasm; and damage to the mitochondria. These damaged cells were rapidly vacated as the sporozoite moved laterally into one or more adjacent intact host cells without entering the lumen. It is suggested that the host cell initially entered from the lumen becomes so severely traumatized that the parasite of necessity enters an adjacent cell as a prelude to further development. Various aspects of host-cell invasion by coccidia and malarial parasites are reviewed.

Evaluation of a gel-immunization technique used with two different Immucox vaccine formulations in battery and floor-pen trials with broiler chickens

Abstract The use of a gel-immunization technique with Immucox vaccination was compared and evaluated against other immunization methods in battery and floor-pen immunization trials. Gel immunization was found to be superior to immunization by gavage, by spray cabinet, or by the conventional delivery method of Immucox in a battery trial. Significantly enhanced protection as measured by weight gain, coupled with the establishment of a more uniform primary immunizing infection as evidenced by greater intestinal lesions and increased oocyst shedding, was seen in gel-immunized birds. In addition, cross-protective battery trials determined that the strain of Eimeria maxima found in the Immucox vaccine failed to elicit protection against a recent field isolate of E. maxima as measured by average weight gain and lesion scores. A reformulation of the Immucox vaccine that included the field isolate of E.␣maxima was required to elicit a protective immune response against challenge by the field strain. A floor-pen experiment demonstrated that gel immunization of 1-day-old roaster chickens resulted in performance parameters of average weight gain, average bird weight, and feed conversion that did not differ significantly from those recorded for medicated nonimmunized birds.

Scanning and transmission electron microscopy of host cell pathology associated with penetration byEimeria papillata sporozoites

Scanning and electron microscopy was used to study the pathogenesis that occurred in mouse epithelial cells that had been penetrated byEimeria papillata sporozoites. Optimal penetration of parasites injected into nonligated and ligated mouse intestine was found to occur at 4–15 min post-inoculation. During initial penetration, the parasite caused disruption of the microvilli of the intestinal cells, which led to detachment of the microvilli from the plasma membrane of the penetrated cell. Host cells penetrated by the parasite showed extensive destruction of the internal cellular organization together with blebbing of host-cell cytoplasm and release of internal organelles such as mitochondria. Ultimately, the penetrated cells completely broke down, leaving vacuolated areas next to ultrastructurally normal epithelial cells.

The effect of high n-3 fatty acids diets on the ultrastructural development of Eimeria tenella

A study of development of Eimeriatenella in chickens fed high n-3 fatty acids (n-3FA) diets showed ultrastructural degeneration of both asexual and sexual parasite stages. Abnormal shedding of asexual and sexual parasite developmental stages into the cecal lumen was also observed. Ultrastructural degeneration was characterized by cytoplasmic vacuolization, chromatin condensation within the nucleus, a lack of parasitophorous vacuole delineation, and, in some cases, a complete loss of parasite ultrastructural organization. The results of this study indicate that diets high in n-3FA may be useful in the control of avian coccidia.

The effect of dietary protein level on performance characteristics of coccidiosis vaccinated and nonvaccinated broilers following mixed-species Eimeria challenge

A series of experiments were conducted to investigate the effect of starter diet protein levels on the performance of broilers vaccinated with a commercially available live oocyst coccidiosis vaccine before subsequent challenge with a mixed-species Eimeria challenge. Data indicated that an increasing protein concentration in the starter diet improved broiler performance during coccidiosis vaccination. Prechallenge performance data indicated that vaccination could decrease BW and increase feed conversion ratio. The time period most important for the observed effects appeared to be between 13 and 17 d of age. This reduction in performance parameters of vaccinated broilers compared with nonvaccinated broilers was eliminated by the conclusion of the experiments (27 d) in the diet groups with higher protein. Vaccination was effective at generating protective immunity against Eimeria challenge, as evidenced by increased (P < 0.05) BW gain, improved feed conversion, reduced postchallenge mortality, and reduced lesion development in vaccinated broilers compared with nonvaccinated broilers. These observations support numerous other reports that confirm live oocyst vaccination can be used effectively as a preventive against avian coccidiosis in commercially reared broilers. More important, these findings suggest that reduced protein concentration of starter diets can lead to significant losses in broiler performance when using a vaccination program to prevent coccidiosis.

Evaluation of the effect of peptidyl membrane-interactive molecules on avian coccidia

Abstract This study examined the lytic effect of seven different synthetic peptidyl membrane-interactive molecules (Peptidyl-MIMs) on sporozoites of five different species of Eimeria infecting chickens and merozoites of two different species that infect chickens. All Peptidyl-MIMs (pMIMs) demonstrated antiparasitic effects at concentrations of 1–50 μM during incubation periods varying from 1 to 20 min. In addition, electron microscopy showed that ultrastructural degeneration of the pellicle of sporozoite stages of the parasites occurred within 5–10 min of exposure to 5-μM concentrations of three different pMIMs. Pore-like openings were seen in the pellicle of the sporozoites at the ultrastructural level, which indicated that the pMIMs had the same mechanism of action on the parasites as that reported from studies done on bacteria. A reduction in lesion scores was seen in chickens treated orally with 10-, 50-, or ␣␣␣␣␣75-μM concentrations of two different proteolytic stabilized (methylated) pMIMs after challenge with three different species of avian coccidia in battery-cage trials. Collectively these data indicate that pMIMs may be useful in the control of coccidiosis in poultry.

Ultrastructural observations of development ofEimeria tenella in a novel established avian-derived cell line

Transmission electron microscopy was used to study the in vitro development ofEimeria tenella in a novel established avian-derived cell line (designated CEV-1/F7) used for antigen production in chicken immunization studies. Sporozoites ofE. tenella were inoculated onto cell monolayers and the cells were fixed at 24-h intervals. Large numbers of intracellular sporozoites were seen at 24 h postinoculation (p.i.), and trophozoites were identified at 24–48 h p.i. Immature schizonts, some with budding merozoites, were seen by 48 h p.i. At 72–96 h p.i., immature and mature schizonts and extracellular merozoites were observed. No merozoite invasion occurred, but immature second-generation schizogony was seen in parasitophorous vacuoles of first-generation schizonts. No further development occurred and degeneration of most schizonts was seen by 120–144 h p.i. The results confirmed synchronous development ofE. tenella until 48 h p.i., followed by asynchronous development and ultrastructural degeneration with increased incubation time.

Biotechnology in perspective

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