Molecular characterization of cDNA coding for 33.5 and 41 kDa oocyst and sporocyst proteins that are differentially regulated in different strains of Eimeria maxima.

Eimeria maxima (APU1 and APU2) differ in virulence for chickens, due in part to the greater fecundity of the former. In a previous study, RNA-seq was used to identify a transcripts upregulated in E. maxima APU1 compared to E. maxima APU2. In this study, 2 of these upregulated genes (EMWEY 23530 and EMWEY 48910) were characterized by first confirming upregulation using quantitative RT-PCR. For both EMWEY 23530 and EMWEY 48910, RNA transcription was fairly consistent during sporulation. The extent of differential expression was about 2-fold log2 higher in APU-1 compared to APU-2 (peaking at 18 h for EMWEY 23530 and 0 h for EMWEY 48910). EMWEY 23530 and EMWEY 48910 cDNA were cloned and expressed as polyHis-fusion proteins in Escherichia coli. The observed size of recombinant EMWEY 23530 was 24 kDa; the observed size of recombinant EMWEY 48910 was 35 kDa, which are consistent with the predicted size based on the coding sequences. Immunostaining 2D gel blots of E. maxima APU1 and APU2 oocyst/sporocyst protein with antisera specific for EMWEY 23530 identified a 33.5 kDa protein with a pH 7.4 isoelectric point (Emax p33.5). Similar 2D gel blot analysis with EMWEY 48910 identified a 41 kDa protein with a pH 7.2 isoelectric point (Emax p41). The intensity of Emax p33.5 and Emax p41 was noticeably greater in oocyst/sporocyst proteins from E. maxima APU1 compared to E. maxima APU2. This was corroborated by ELISA wherein equal amounts of total E. maxima APU1 and APU2 protein were probed with serial dilutions of anti-rEmax p33.5 or anti-rEmax p41. Immunofluorescence (IFA) staining of permeabilized unsporulated E. maxima APU1 and APU2 oocysts revealed Emax p33.5 to be localized in one end of oocysts, while Emax p41 appeared on the surface of oocysts. After sporulation, the p33.5 and p41 antigens appeared loosely associated with sporocysts. Taken together, these data confirm excess expression of two proteins in the E. maxima strain characterized by greater fecundity and virulence, and may provide insight into basis for phenotypic differences among different E. maxima.

A Study of Cross-Protection between Eimeria maxima Immunovariants.

For reasons unknown, Eimeria maxima is unique among Eimeria species infecting chickens in the immunovariability it displays among isolates from different geographical areas. Eimeria maxima oocysts (named EmaxAPU3) were isolated late in grow-out (6 weeks) from litter in a commercial broiler operation that was using Eimeria vaccination as the coccidiosis control program. Cross-protection studies (n = 4) were conducted in immunologically naïve chickens between EmaxAPU3 and two E. maxima lab strains (EmaxAPU1, EmaxAPU2) by immunizing with one E. maxima strain and challenging with either the homologous or heterologous E. maxima. As measured by oocyst output, immunization with EmaxAPU1 protected against homologous challenge (EmaxAPU1) and against heterologous challenge with EmaxAPU3, but not against EmaxAPU2. Similarly, immunization with EmaxAPU3 protected against homologous challenge (EmaxAPU3) and against heterologous challenge with EmaxAPU1, but not against EmaxAPU2. Immunization of chickens with EmaxAPU2 elicited a protective response against homologous challenge (EmaxAPU2), but not against EmaxAPU1 nor EmaxAPU3. The most plausible explanation for the appearance of this immunovariant late in grow-out is that E. maxima APU3 escaped immunity directed to E. maxima antigenic types in the commercial vaccine.

Chromosomal scale assembly reveals localized structural variants in avian caecal coccidian parasite Eimeria tenella.

Eimeria tenella is a major cause of caecal coccidiosis in commercial poultry chickens worldwide. Here, we report chromosomal scale assembly of Eimeria tenella strain APU2, a strain isolated from commercial broiler chickens in the U.S. We obtained 100× sequencing Oxford Nanopore Technology (ONT) and more than 800× Coverage of Illumina Next-Seq. We created the assembly using the hybrid approach implemented in MaSuRCA, achieving a contiguous 51.34 Mb chromosomal-scale scaffolding enabling identification of structural variations. The AUGUSTUS pipeline predicted 8060 genes, and BUSCO deemed the genomes 99% complete; 6278 (78%) genes were annotated with Pfam domains, and 1395 genes were assigned GO-terms. Comparing E. tenella strains (APU2, US isolate and Houghton, UK isolate) derived Houghton strain of E. tenella revealed 62,905 high stringency differences, of which 45,322 are single nucleotide polymorphisms (SNPs) (0.088%). The rate of transitions/transversions among the SNPs are 1.63 ts/tv. The strains possess conserved gene order but have profound sequence heterogeneity in a several chromosomal segments (chr 2, 11 and 15). Genic and intergenic variation in defined gene families was evaluated between the two strains to possibly identify sequences under selection. The average genic nucleotide diversity of 2.8 with average 2 kb gene length (0.145%) at genic level. We examined population structure using available E. tenella sequences in NCBI, revealing that the two E. tenella isolates from the U.S. (E. tenella APU2 and Wisconsin, "ERR296879") share a common maternal inheritance with the E. tenella Houghton. Our chromosomal level assembly promotes insight into Eimeria biology and evolution, hastening drug discovery and vaccine development.

Relationship between Eimeria oocyst infectivity for chickens and in vitro excystation of E. acervulina, E. maxima, and E. tenella oocyst during long-term storage.

Vaccination of chickens against avian coccidiosis in chickens often involves storing Eimeria oocysts for months after oocyst propagation and sporulation. The purpose of this study was to determine how long E. acervulina, E. maxima, and E. tenella oocysts remained viable when stored at refrigeration (4°C) or egg room (20°C) temperatures. Separate tubes containing E. acervulina, E. maxima, or E. tenella oocysts were stored at these temperatures and a sample removed every 3 mo for inoculating chickens for evidence of a patent infection. Also, an aliquot of each Eimeria species at each time-temperature combination was subjected to in vitro excystation to quantify the relative number of released sporozoites to intact (nonexcysted) sporocysts. Eimeria tenella appeared to be most susceptible to storage in that no oocyst production was observed at 9 mo at either temperature. Although E. maxima oocysts were viable at 9 mo, no oocyst production was observed at 12 mo storage at these 2 temperatures. Quite unexpected was that E. acervulina was much more stable than E. tenella and E. maxima remaining viable up to and including 27 mo at 4°C and up to and including 12 mo at 20°C. No consistent correlation was observed between in vivo oocyst production and in vitro excystation arising from these 2 respective temperatures (E. acervulina r = 0.58, r = 0.54; E. maxima r = 0.90, r = 0.54; E. tenella r = 0.38, r = 0.90). These data indicate that attention must be paid to time and temperature of Eimeria oocyst storage, and that sporozoite excystation may not be a good indicator of oocyst viability, particularly at later timepoints in incubation.

Effects of codon optimization on expression in Escherichia coli of protein-coding DNA sequences from the protozoan Eimeria.

The objective of this study was to compare the levels of recombinant protein from three Eimeria genes before and after optimization of codons for expression in Escherichia coli. Protein coding sequences from Eimeria maxima (EmaxSO7, EmaxIMP1) and Eimeria acervulina (EAH00033530) were cloned with or without prior codon optimization and expressed as polyHis fusion proteins. All three outcomes: higher, lower, or no change in the yield of amount of recombinant protein were observed suggesting that codon optimization alone for expression in E. coli does not inevitably lead to higher expression levels. Analysis of codon usage for each gene sequence revealed that, similar to other organisms, Eimeria intersperses rare and frequently used codons in protein-coding sequences. However, no relationship was observed between the predicted protein structure and the location of major and minor codons, suggesting that codon selection in this apicomplexan parasite is influenced by factors other than regional secondary protein structure.

Polymerase Chain Reaction Directed to Eimeria ITS1 rDNA or a Single-Copy Orthologue Corroborates Standard Micro-oocyst Analysis of Intestinal Tissue from Chickens Infected with E. acervulina, E. maxima, or E. tenella.

The purpose of this study was to compare micro-oocyst counts of Eimeria to PCR analysis of intestinal DNA from smears of duodenum, jejunum/ileum, and cecum of chickens infected with Eimeria acervulina, Eimeria maxima, or Eimeria tenella oocysts. Broiler chicks were infected in triplicate with various doses of E. acervulina, E. maxima, or E. tenella oocysts and were necropsied 5-6 days later to recover duodenal, jejunal, or cecal tissue for micro-oocyst count and for DNA recovery. Micro-oocyst counts were done independently by three individuals. Micro-oocyst counts and PCR directed to ITS1 rDNA or to a single-copy orthologue (SCO 5995) displayed a linear relationship with oocyst dose for each Eimeria species. A strong correlation was found between mean micro-oocyst counts and both PCR assays for E. acervulina (r = 0.78-0.94), E. maxima (r = 0.79-0.91), and E. tenella (r = 0.85-0.96). There was good agreement between ITS1 and SCO 5995 PCR assays: E. acervulina (r = 0.92), E. maxima (r = 0.79), and E. tenella (r = 0.93). However, only ITS1 PCR analysis corroborated micro-oocyst counts of Eimeria oocyst DNA recovered from Eimeria-infected broiler chickens submitted to a poultry diagnostic laboratory. These findings suggest that ITS1 PCR or SCO PCR can validate traditional micro-oocyst counts used in quantifying Eimeria infection in chickens. Additional studies may provide a method for estimating the relative abundance of each Eimeria species in a natural infection.

Reacción en cadena de la polimerasa dirigida al gene ITS1 rDNA de Eimeria o a un ortólogo de copia única corrobora el análisis estándar de microquistes del tejido intestinal de pollos infectados con E. acervulina, E. maxima o E. tenella. El propósito de este estudio fue comparar los recuentos de micro-ooquistes de Eimeria con el análisis PCR del ADN intestinal de frotis de duodeno, yeyuno/íleon y ciego de pollos infectados con ooquistes de Eimeria acervulina, Eimeria maxima o Eimeria tenella. Los pollos de engorde se infectaron por triplicado con varias dosis de ooquistes de E. acervulina, E. maxima o E. tenella y se les realizó la necropsia entre los cinco y seis días después para recuperar tejido duodenal, yeyunal o cecal para el recuento de micro-ooquistes y para la recuperación de ADN. Los recuentos de micro-ooquistes se realizaron de forma independiente por tres personas. Los recuentos de micro-ooquistes y la PCR dirigida al gene ITS1-rDNA o a un ortólogo de copia única (SCO 5995) mostraron una relación lineal con la dosis de ooquistes para cada especie de Eimeria. Se encontró una fuerte correlación entre el recuento medio de micro-ooquistes y ambos ensayos de PCR para E. acervulina (r = 0.78­0.94), E. maxima (r = 0.79­0.91) y E. tenella (r = 0.85­0.96). Hubo una buena concordancia entre los ensayos de PCR ITS1 y SCO 5995: E. acervulina (r = 0.92), E. maxima (r = 0.79) y E. tenella (r = 0.93). Sin embargo, solo el análisis ITS1 PCR corroboró los recuentos de micro-ooquistes de ADN de ooquistes de Eimeria recuperados de pollos de engorde infectados con Eimeria enviados a un laboratorio de diagnóstico avícola. Estos hallazgos sugieren que los métodos de ITS1 PCR o SCO PCR pueden validar los recuentos tradicionales de micro-ooquistes utilizados para cuantificar la infección por Eimeria en pollos. Estudios adicionales pueden proporcionar un método para estimar la abundancia relativa de cada especie de Eimeria en una infección natural.

Cloning and expression of a cDNA coding for Eimeria acervulina 25 kDa protein associated with oocyst and sporocyst walls.

The purpose of this study was to characterize a gene named EAH 00033530 identified by RNAseq analysis of sporulating Eimeria acervulina oocysts and its encoded protein. Quantitative RT-PCR analysis revealed peak expression of EAH 00033530 mRNA early (3-6 h) in sporulation followed by downregulation at 12-24 h. The gene for EAH 00033530 was expressed in Escherichia coli as a 70 kDa polyHis fusion protein (rEAH 00033530). Antisera prepared against rEAH 00033530 protein identified in immunoblotting a native 25 kDa E. acervulina protein (Ea25) that was present in oocyst-sporocyst extracts after treatment with the reducing agent DTT. Immunofluorescence staining using anti-rEa25 localized the protein to both E. acervulina oocyst and sporocyst walls, but not to sporozoites. The protein may be produced during in vivo oocyst development because immunostaining of duodenal tissue from E. acervulina-infected chickens revealed oocyst wall expression. As observed by ELISA, rEa25 protein appears to elicit a humoral immune response in chickens infected with non-irradiated or radiation-attenuated E. acervulina oocysts.

Metagenomic Analysis of 16S Clostridium perfringens Amplicons Corroborates C. perfringens Counts on Select Agar and C. perfringens PCR Analyses of Bacteria in Broiler Farm Litter.

The purpose of this study was twofold-first, to determine whether analysis of bacterial 16S ribosomal RNA (rRNA) in poultry litter corroborated standard Clostridium perfringens counts and PCR assay, and second, to find whether a correlation between 16S rRNA analysis and netB or Tpel toxin PCR intensity with chick mortality existed. At three time points of growout (0, 2, and 4 wk) litter samples were collected from 23 broiler houses representing eight farms during a coccidiosis vaccine control program. DNA extracted from these samples was used for microbiota determination by sequencing the hypervariable V3-V4 region of bacterial 16s rRNA. Obtained sequences were analyzed by QIIME 2 and the Greengenes database for taxonomic composition and relative abundance of C. perfringens in the litter bacterial population. Clostridium perfringens counts on select agar and semiquantitative PCR for C. perfringens were compared with 16S analysis for equivalence testing. Relative abundance of C. perfringens estimated by 16S analysis and semiquantitative PCR for netB and Tpel toxin DNA were analyzed by Pearson linear correlation and statistical equivalence analyses with cumulative chick mortality at 4 and 9 wk growout. When data from all time points were combined, abundance estimates by C. perfringens 16S were statistically equivalent (α = 0.10) to both C. perfringens PCR and C. perfringens counts. Yet, no correlations were observed between any estimate of C. perfringens abundance and cumulative percent chick mortality at 4 or 9 wk growout. However, correlation analyses revealed a significant linear relationship between netB signal at 0 wk (r = 0.55) and 4 wk (r = 0.46) and cumulative mortality at 9 wk growout (P < 0.05). Similarly, abundance of Tpel at 0 and 2 wk showed a linear relationship with cumulative percent mortality at both 4 and 9 wk growout (0.44 ≤ r ≤ 0.54, P < 0.05). No correlations were observed between any other genera or species determined by 16S and cumulative percent chick mortality.

Correlation Between Clostridium perfringens Alpha- and NetB-Toxin and Chick Mortality in Commercial Broiler Farms During Different Anticoccidial Control Programs.

The purpose of the present study was to determine whether a correlation existed between chick mortality and the presence of Clostridium perfringens alpha-toxin and NetB-toxin genes (cpa and netB) in C. perfringens recovered from litter in commercial broiler houses. Because coccidiosis predisposes chickens to necrotic enteritis, the concentration of Eimeria oocysts in these samples was measured, and the numbers were used in similar correlation analyses. Litter samples were collected at 0, 2, and 4 wk growout from six broiler farms (18 houses total) during an anticoccidial drug (ACD) control program and from nine broiler farms (23 houses total) during an Eimeria vaccine (VAC) control program. Of these, litter samples were collected from five farms during both ACD and VAC programs. The litter samples were processed for Eimeria oocyst and C. perfringens spore enumerations by standard parasitologic and microbiologic techniques. DNA was also extracted for C. perfringens DNA for PCR detection of genes coding for alpha- and NetB-toxin. A general trend during the ACD programs was a transient decrease in both Eimeria maxima and non-E. maxima (Eamipt) numbers at 2 wk growout. The pattern was slightly different during VAC with E. maxima and Eamipt levels increasing over time. Average concentrations of C. perfringens in litter were highest at 2 wk (∼105-106 spores/g) during ACD and at placement during VAC (∼105-106 spores/g). During the ACD program, a strong correlation was observed between 0 and 3-wk chick mortality and the presence at placement (0 wk) of netB (r = 0.42-0.48) or cpa (r = 0.55-0.67). A very strong correlation was observed in 0-5-wk chick mortality and the presence of netB at 4 wk growout (0.73-0.95). During a VAC program, a strong correlation was only observed between the presence of netB at placement and 0-1-wk chick mortality (r = 0.67).

Excystation of Eimeria acervulina, E. maxima, and E. tenella differs in response to trypsin and chymotrypsin and the presence of reducing agents DTT and TCEP.

Release of sporozoites from Eimeria oocysts/sporocysts is an essential step in the intracellular development of the parasite in its host. Little is known about this process except that elevated temperature (∼ 40 °C) plus trypsin and bile salts are required for sporozoite to escape from sporocysts. In this study, it was found that adding a reducing agent, either dithiothreitol (DTT) or Tris(2-carboxyethyl)phosphine hydrochloride (TCEP), increased the lifespan of sporozoites released from Eimeria maxima. While the addition of DTT or TCEP affected the apparent molecular weight of trypsin, it did not interfere with excystation of E. maxima, but rather had a positive effect on the number of viable sporozoites present after release. This effect was time-dependent in that the number of intact sporozoites at 15 and 30 min after excystation was similar between untreated and DTT- or TCEP-treated sporocysts. However, by 45-60 min, virtually no sporozoites were observed in excystation fluid not containing DTT or TCEP. Of interest is that this effect appeared to be Eimeria species-dependent. Eimeria acervulina and E. tenella sporozoites remained viable for at least 60 min after excystation in the absence of DTT or TCEP. The effect of DTT and TCEP on chymotrypsin was also studied with all 3 Eimeria species because there is some evidence that chymotrypsin is an effective excystation enzyme. Indeed, E. maxima sporozoites excysting from sporocysts with chymotrypsin in the presence of DTT or TCEP remained viable for at least 60 min after release, unlike excystation done in the absence of these reducing agents. Chymotrypsin was capable of excysting E. acervulina in the presence or absence of DTT or TCEP. Of interest, is that chymotrypsin was ineffective in the excystation of E. tenella. These findings suggest that trypsin and chymotrypsin have differential effects on sporozoite excystation and that reducing agents may alter sites on the enzyme that affect sporozoite viability, but not release from sporocysts.

Incorporation of a recombinant Eimeria maxima IMP1 antigen into nanoparticles confers protective immunity against E. Maxima challenge infection.

The purpose of this study was to determine if conjugating a recombinant Eimeria maxima protein, namely EmaxIMP1, into 20 nm polystyrene nanoparticles (NP) could improve the level of protective immunity against E. maxima challenge infection. Recombinant EmaxIMP1 was expressed in Escherichia coli as a poly-His fusion protein, purified by NiNTA chromatography, and conjugated to 20 nm polystyrene NP (NP-EmaxIMP1). NP-EMaxIMP1 or control non-recombinant (NP-NR) protein were delivered per os to newly-hatched broiler chicks with subsequent booster immunizations at 3 and 21 days of age. In battery cage studies (n = 4), chickens immunized with NP-EMaxIMP1 displayed complete protection as measured by weight gain (WG) against E. maxima challenge compared to chickens immunized with NP-NR. WG in the NP-EMaxIMP1-immunized groups was identical to WG in chickens that were not infected with E. maxima infected chickens. In floor pen studies (n = 2), chickens immunized with NP-EMaxIMP1 displayed partial protection as measured by WG against E. maxima challenge compared to chickens immunized with NP-NR. In order to understand the basis for immune stimulation, newly-hatched chicks were inoculated per os with NP-EMaxIMP1 or NP-NR protein, and the small intestine, bursa, and spleen, were examined for NP localization at 1 h and 6 h post-inoculation. Within 1 h, both NP-EMaxIMP1 and NP-NR were observed in all 3 tissues. An increase was observed in the level of NP-EmaxIMP1 and NP-NR in all tissues at 6 h post-inoculation. These data indicate that 20 nm NP-EmaxIMP1 or NP-NR reached deeper tissues within hours of oral inoculation and elicited complete to partial immunity against E. maxima challenge infection.

Eimeria Oocyst Concentrations and Species Composition in Litter from Commercial Broiler Farms During Anticoccidial Drug or Live Eimeria Oocyst Vaccine Control Programs.

The purpose of this study was to determine if Eimeria oocyst concentrations and species composition in commercial broiler house litter changed during different cycles of anticoccidial drug (ACD) or live Eimeria oocyst vaccine (VAC) control programs and if there was a correlation between Eimeria oocyst levels and broiler performance. Litter samples were collected from a total of 15 different broiler farms encompassing a total of 45 individual houses during at least one complete grow-out cycle over a 21-mo period. Of these 15 broiler farms, three were followed for the entire 21-mo period spanning three ACD and four VAC cycles. Samples were collected at 2, 4, and 7-8 wk of grow-out corresponding to starter, grower, and withdraw periods of the ACD cycle. On a number of occasions, litter samples were obtained just prior to chick placement. Eimeria oocysts were isolated from all samples, counted by microscopy, and extracted for DNA to identify Eimeria species by ITS1 PCR. In general, Eimeria oocyst concentration in litter reached peak levels at 2-4 wk of grow-out regardless of coccidiosis control measure being used. However, peak oocyst numbers were sometimes delayed until 7-8 wk, indicating some level of Eimeria spp. drug resistance or incomplete vaccine coverage. Eimeria maxima , Eimeria acervulina , Eimeria praecox, and Eimeria tenella were generally present in all samples, and no difference in the species composition was noted between houses on a particular farm. While Eimeria species composition was similar among houses, Eimeria spp. oocyst levels exhibited sporadic peaks in one house of a given location's houses. Of particular interest was the observed correlation between E. maxima oocyst abundance and chick mortality. However, no correlation was observed in E. maxima oocyst levels, and the performance parameters adjusted feed conversion ratio and average daily weight gain. This study showed that understanding the dynamics of Eimeria spp. oocyst levels and species composition in litter during ACD or VAC programs may provide insight into the effectiveness of coccidiosis control measures in commercial broiler production.

Protecting chickens against coccidiosis in floor pens by administering Eimeria oocysts using gel beads or spray vaccination.

Control of avian coccidiosis is increasingly being achieved by the administration of low doses of Eimeria oocysts to newly hatched chicks. The purpose of this study was to test the efficacy of gel beads containing a mixture of Eimeria acervulina, Eimeria maxima, and Eimeria tenella oocysts as a vaccine to protect broilers raised in contact with litter. Newly hatched chicks were either sprayed with an aqueous suspension of Eimeria oocysts or were allowed to ingest feed containing Eimeria oocysts-incorporated gel beads. Control, 1-day-old chicks were given an equivalent number of Eimeria oocysts (10(3) total) by oral gavage or received no vaccine (nonimmunized controls). All chicks were raised in floor-pen cages in direct contact with litter. At 4 wk of age, all chickens and a control nonimmunized group received a high-dose E. acervulina, E. maxima, and E. tenella challenge infection. Chickens immunized with Eimeria oocysts in gel beads or by spray vaccination displayed significantly (P < 0.05) greater weight gain (WG) compared to nonimmunized controls. Feed conversion ratio (FCR) also showed a significant (P < 0.05) improvement in both groups relative to nonimmunized controls. Moreover, WG and FCR in both groups was not significantly different (P > 0.05) from chickens immunized by oral gavage or from nonimmunized, noninfected controls. Oocyst excretion after Eimeria challenge by all immunized groups was about 10-fold less than in nonimmunized controls. These findings indicate that immunization efficacy of gel beads and spray vaccination is improved by raising immunized chicks in contact with litter.

Differing susceptibilities of Eimeria acervulina, Eimeria maxima, and Eimeria tenella oocysts to desiccation.

Outbreaks of avian coccidiosis may occur when susceptible chickens are raised on litter containing viable Eimeria oocysts. The purpose of this study was to compare the relative sensitivities of Eimeria acervulina, Eimeria maxima, and Eimeria tenella oocysts to dessication. Sporulated E. acervulina, E. maxima, or E. tenella oocysts were incorporated into gelatin beads and incubated at 32 C for 0, 1, 2, or 3 days. In vitro oocyst excystation rates were measured for each combination of Eimeria species and incubation time. Day-old broiler chicks were allowed to ingest the oocysts-containing beads, and total oocyst production was measured from days 5-8 post-inoculation. Although no effect on excystation was observed, E. maxima oocysts displayed greater resistance to drying compared to E. acervulina and E. tenella oocysts. Eimeria acervulina oocyst production decreased 100-fold after 1-2 days incubation. Eimeria tenella oocysts were slightly more resistant to drying in that a 100-fold decrease in oocyst production was delayed until 2 days. For both E. acervulina and E. tenella , very few oocysts were observed after 3 days incubation. Eimeria maxima oocyst production remained high at all time points. Subsequent studies revealed E. maxima oocyst production was ablated only after 5 days incubation. These findings may explain in part the observed prevalence of E. maxima in litter from commercial poultry operations.

Gel-Bead Delivery of Eimeria oocysts protects chickens against coccidiosis.

Vaccines composed of either virulent or attenuated Eimeria spp. oocysts have been developed as an alternative to medication of feed with ionophore drugs or synthetic chemicals. The purpose of this study was to evaluate the use of gel-beads containing a mixture of Eimeria acervulina, Eimeria maxima, and Eimeria tenella oocysts as a vaccine against coccidiosis. Newly hatched chicks (Gallus gallus domesticus) were either sprayed with an aqueous suspension of Eimeria oocysts or were allowed to ingest feed containing Eimeria oocysts-incorporated gel-beads. Control day-old chicks were given an equivalent number of Eimeria oocysts (10(4) total) by oral gavage. After 3 days, chicks were randomly assigned to individual cages, and feces were collected between days 5 and 8 postinfection. All samples were processed for total Eimeria oocysts. At 4 wk of age, all chickens and a control nonimmunized group received a high-dose E acervulina, E maxima, and E. tenella challenge infection. Oocyst excretion by chicks fed gel-beads or inoculated by oral gavage was 10- to 100-fold greater than that of chicks spray-vaccinated with the Eimeria oocysts mixture (log 6.3-6.6 vs. log 4.8). Subsequent protection against challenge as measured by weight gain and feed conversion efficiency was significantly greater (P < 0.05) in gel-bead and oral gavage groups compared with spray-vaccinated or nonimmunized groups. Also, gel-bead and oral gavage groups showed no significant difference (P > 0.05) in weight gain and feed conversion efficiency compared with nonchallenged controls. These findings indicate that incorporation of Eimeria spp. oocysts in gel-beads may represent an effective way to deliver live oocyst vaccines to day-old chicks for preventing subsequent outbreaks of coccidiosis in the field.

Impact of A Smoking Prevention Interactive Experience (ASPIRE), an interactive, multimedia smoking prevention and cessation curriculum for culturally diverse high-school students.

Few studies have examined the long-term efficacy of computer-based smoking prevention and cessation programs. We analyzed the long-term impact of A Smoking Prevention Interactive Experience (ASPIRE), a theoretically sound computer-based smoking prevention and cessation curriculum for high school students. Sixteen predominantly minority, inner-city high schools were randomly assigned to receive the ASPIRE curriculum or standard care (receipt of the National Cancer Institute's Clearing the Air self-help booklet). A total of 1160 students, 1098 of whom were nonsmokers and 62 smokers at baseline, were included. At 18-month follow-up, among baseline nonsmokers, smoking initiation rates were significantly lower in the ASPIRE condition (1.9% vs. 5.8%, p < .05). Students receiving ASPIRE also demonstrated significantly higher decisional balance against smoking and decreased temptations to smoke. Differences between groups in self-efficacy and resistance skills were not significant. There was a nonsignificant trend toward improved smoking cessation with ASPIRE, but low recruitment of smokers precluded conclusions with respect to cessation. ASPIRE demonstrated the potential for an interactive multimedia program to promote smoking prevention. Further studies are required to determine ASPIRE's effects on cessation.

Aging, gender and APOE isotype modulate metabolism of Alzheimer's Abeta peptides and F-isoprostanes in the absence of detectable amyloid deposits.

Aging and apolipoprotein E (APOE) isoform are among the most consistent risks for the development of Alzheimer's disease (AD). Metabolic factors that modulate risk have been elusive, though oxidative reactions and their by-products have been implicated in human AD and in transgenic mice with overt histological amyloidosis. We investigated the relationship between the levels of endogenous murine amyloid beta (Abeta) peptides and the levels of a marker of oxidation in mice that never develop histological amyloidosis [i.e. APOE knockout (KO) mice with or without transgenic human APOEepsilon3 or human APOEepsilon4 alleles]. Aging-, gender-, and APOE-genotype-dependent changes were observed for endogenous mouse brain Abeta40 and Abeta42 peptides. Levels of the oxidized lipid F2-isoprostane (F2-isoPs) in the brains of the same animals as those used for the Abeta analyses revealed aging- and gender-dependent changes in APOE KO and in human APOEepsilon4 transgenic KO mice. Human APOEepsilon3 transgenic KO mice did not exhibit aging- or gender-dependent increases in F2-isoPs. In general, the changes in the levels of brain F2-isoPs in mice according to age, gender, and APOE genotype mirrored the changes in brain Abeta levels, which, in turn, paralleled known trends in the risk for human AD. These data indicate that there exists an aging-dependent, APOE-genotype-sensitive rise in murine brain Abeta levels despite the apparent inability of the peptide to form histologically detectable amyloid. Human APOEepsilon3, but not human APOEepsilon4, can apparently prevent the aging-dependent rise in murine brain Abeta levels, consistent with the relative risk for AD associated with these genotypes. The fidelity of the brain Abeta/F2-isoP relationship across multiple relevant variables supports the hypothesis that oxidized lipids play a role in AD pathogenesis, as has been suggested by recent evidence that F2-isoPs can stimulate Abeta generation and aggregation.

Inclusion of CpG adjuvant with plasmid DNA coding for NcGRA7 improves protection against congenital neosporosis.

The present study showed that incorporation of CpG adjuvant into plasmid DNA coding for NcGRA7 antigen resulted in a twofold increase in the level of protection against congenital transfer of Neospora caninum. The level of protection was considerably higher than that observed in pups born from dams immunized with nonrecombinant plasmid.

Immunization of mice with plasmid DNA coding for NcGRA7 or NcsHSP33 confers partial protection against vertical transmission of Neospora caninum.

The purpose of the present study was to use direct plasmid deoxyribonucleic acid (DNA) injection to identify specific antigens that confer protection against congenital transfer of Neospora caninum. Inbred BALB/c mice were vaccinated before pregnancy with a recombinant plasmid containing sequences encoding N. caninum antigen NcGRA7 or NcsHSP33. The mice were challenged with N. caninum tachyzoites at 10-12 days of gestation. Whereas 100% of pups born from dams immunized with control plasmid contained detectable levels of N. caninum DNA in a Neospora-specific polymerase chain reaction assay, only 46% of pups from pCMVi-NcGRA7-immunized mice and 53% of pCMVi-NcsHSP33-immunized mice were N. caninum positive, and none of the mice immunized with tachyzoite extract contained N. caninum DNA. Thus, immunization of mice with plasmid DNA expressing N. caninum antigens conferred partial protection against congenital neosporosis.

Experimental infection of sheep with Neospora caninum oocysts.

The purpose of the present study was to investigate the potential of Neospora caninum oocysts to infect sheep and determine whether N. caninum DNA could be detected by polymerase chain reaction (PCR) assay in blood and brain of sheep after oocyst inoculation. Six ewes were inoculated per os with 10(4) N. caninum oocysts, whereas 2 ewes served as uninoculated controls. All sheep were bled weekly for 7 wk after inoculation. Blood was analyzed for the presence of N. caninum DNA by 2 different PCR assays, as well as for the presence of antibodies to recombinant and native N. caninum antigens. Neospora caninum DNA was detected in 2 sheep as early as 7 days after oocyst inoculation (DAOI). All 6 sheep were PCR positive by 32 days and remained positive until the end of the study at 49 DAOI. Aside from 1 ewe, all sheep inoculated with N. caninum oocysts contained detectable N. caninum DNA in the brain tissue collected at 49 DAOI. Unlike with PCR, no lesion or parasite was detected by immunohistochemistry. Antibodies were detected by enzyme-linked immunosorbent assay, Neospora agglutination test, or immunoblotting to either native or recombinant N. caninum antigens in sheep inoculated with oocysts.