Vaccination with Neospora caninum-cyclophilin and -profilin confers partial protection against experimental neosporosis-induced abortion in sheep.

The purpose of this study was to evaluate the protective efficacy of a vaccine consisting of recombinant Neospora caninum-cyclophilin (NcCyP) and -profilin (NcPro) in sheep. At 42 d and 21 d prior to mating, adult Dorset ewes were immunized with the rNcCyP-rNcPro vaccine (Group 1) or co-purifying non-recombinant (NR) control vaccine (Group 2). At 90 days post-mating, all immunized ewes and were challenged by intravenous injection with 106Nesopora caninum Illinois tachyzoites (NcTZ). Significant protection (P < 0.05) was observed in Group 1 with 9 out of 13 ewes giving birth to live-born lambs (69.2%), whereas all Group 2 ewes aborted (6/6). Neospora caninum was detected by PCR in both fetal and placental tissues from all Group 2 aborting ewes and in the placental tissues of Group 1 aborting ewes. In contrast, tissues and placentas of Group 1 live-born lambs were Neospora DNA-negative. Immunoreactive Neospora antigens were demonstrated in placentas associated with abortions, but not in tissues of aborted fetuses or those of the live-born lambs and their associated placentas. Anti-NcCyP and anti-NcPro titers were high in sera from Group 1 ewes and were further boosted by challenge infection, resulting in long-lasting (≥14.5 mos.) elevated titers. Lambs born to Group 1 ewes also had high NcCyP and NcPro titers in pre-colostrum sera. Immunofluorescence staining (IFA) of NcTZ with Group 1 post-immunization sera revealed both surface and internal TZ staining, a pattern consistent with that observed with rabbit sera to rNcCyP or rNcPro. Infection of NR-vaccinated ewes produced high but transient anti-NcCyP and anti-NcPro Ab titers. The results indicate that the NcCyP-NcPro vaccine elicited strong anti-N. caninum responses and conferred significant protection against abortion and transplacental transmission of N. caninum TZ in sheep.

Expression of amino acid and sugar transporters, aminopeptidase, and the di- and tri-peptide transporter PepT1; differences between modern fast growing broilers and broilers not selected for rapid growth.

Within the last 60 yr genetics of broilers have changed to produce rapid growing birds that achieve market weight in 6 wk or less. To investigate the differences in factors that play a role in nutrient processing and uptake between modern fast growing (Ross) and slow growing broilers not selected for growth (ACRBC), a study was carried comparing the expression of 13 genes that encode amino acid transporters (ASCT1, ATBo,+, BoAT, bo, +AT, CAT1, CAT2, EAAT3, γ+LAT1, and LAT1) and sugar transporters (GLUT2 and GLUT5), as well as aminopeptidase (APN) and the di- and tri-peptide transporter PepT1. The growth rate of Ross birds was approximately 4 times greater than that of ACRBCs, and the feed conversion ratio (FCR) was greater in ACRBCs at all-time points measured. Gene expression in the duodenum, jejunum, and ileum was measured at 1, 3, 5, 10, and 14 d post hatch (PH). The expression of genes that encode proteins (particularly ASCT1, ATBo, +, and BoAT) located at the brush border of the gut epithelium was generally higher in ACRBCs especially at earlier time points. The expression of genes that encode proteins located at the basolateral surface of the gut epithelium was less affected. The expression of GLUT2 and GLUT5 was significantly decreased in ACRBCs at most time points and gut segments. Based on the present data we conclude that expression of brush border and sugar transporters in the small intestine can be correlated with growth. Presented increases the identification of the factors that influence growth and will assist future studies of the function of these molecules.

Neospora caninum cytoplasmic dynein LC8 light chain 2 (NcDYNLL2) is differentially produced by pathogenically distinct isolates and regulates the host immune response.

Neospora caninum is the causative agent of bovine neosporosis. A N. caninum cytoplasmic dynein LC8 light chain (NcDYNLL) protein was characterized in this study. Cytoplasmic dyneins, including DYNLLs, belong to the microtubule minus-end-directed motor proteins and are involved in many cellular processes. Previous microarray studies revealed that NcDYNLL was downregulated in the non-pathogenic clone, Ncts-8, when compared with the wild-type NC1 isolate. The present study showed that DYNLLs from different species are highly conserved (>85% identity), and the NcDYNLL belongs to the DYNLL2 family. NcDYNLL2 and Toxoplasma gondii DYNLL2 have identical amino acid sequences, although they are slightly divergent at the genetic level (89% identity). NcDYNLL2 was cloned and expressed in Escherichia coli and purified. NcDYNLL2 was identified in soluble and insoluble fractions of tachyzoite lysate. As expected, soluble NcDYNLL2 was lower in the Ncts-8 lysate when compared with that of NC1 isolate. NcDYNLL2 release by the tachyzoites was low; however, it was increased when tachyzoites were treated with either calcium ionophore or ethanol. The data indicate that NcDYNLL2 may be actively secreted at low levels, but the secretion was upregulated by agents that also augment microneme protein secretions. Immunostaining of NcDYNLL2 in isolated and intracellular Neospora tachyzoites showed a diffuse distribution pattern. Furthermore, rNcDYNLL2 was internalized by the host immune cells and stimulated tumour necrosis factor-α) and interleukin-12 (IL-12) production by murine dendritic cells. Taken together, these results suggest that NcDYNLL2 is a secretory protein that cross-regulates host immunity.

Temporal pattern changes in duodenal protein tyrosine nitration events in response to Eimeria acervulina infection in chickens.

Intracellular generation of nitric oxide (NO) and superoxide anion (SOA) can result in the formation of 3'-nitrotyrosine proteins (NTp). Nitrated proteins usually are associated with significant perturbation in protein function, apoptosis, autophagy, and cell death. We undertook the present study to establish the temporal dynamics of NTp generation in cytokeratin-18-positive epithelial cells (ETCs) of broiler chickens in response to infection with Eimeria acervulina. Duodenal tissue was harvested from noninfected (NOI) and infected (INF) broilers on days (d) 1, 3, 6, 7, and 10 postinfection (PI) and fixed, embedded, and sectioned for quantitative image analysis, immunohistochemistry with antibodies specific to NTp and the SOA-generating enzyme xanthine oxidase (XO). The pixel density characteristics for NTp and XO representative of ETCs demonstrated that NTp and XO increased in intestinal villi as early as d1 PI (P < 0.05 vs. NOI). Progressive increases in NTp were evident in ETCs through d6 PI. For XO, increases in cell content increased only through d3. On d6 and d7 PI, high levels of NTp were present in immune infiltrating cells (IIC) where no XO was detected. The increases in ETC NTp occurred in a defined pattern, significant by villus-to-crypt location for day of infection, initiating in the distal villus and progressing down into the crypts. Two NTp patterns were observed for ETCs: a high level associated with ETCs harboring parasites and a low-level increase in ETCs not containing Eimeria but in proximity to such. The data suggest that NTp and XO responses may mediate some of the processes through which ETCs respond to Eimeria to limit the extent of infection by this pathogen.

The effect of Eimeria maxima infection on the expression of amino acid and sugar transporters aminopeptidase, as well as the di- and tri-peptide transporter PepT1, is not solely due to decreased feed intake.

Coccidiosis caused by Eimeria in poultry is endemic to poultry operations and results in decreased feed intake, diarrhea, and decreased weight gain. The goal was to determine the effect of Eimeria maxima infection on the expression of genes that encode peptide and amino acid transporters (AATs), and also to determine whether decreased feed intake contributes to the change in gene expression by including a pair-fed group of broilers. Three groups of male Ross broilers: 1) not infected, 2) infected, and 3) not infected pair-fed groups were used. Chicks were infected with 1,000 oocysts of E. maxima at 21 d of age. Feed consumption was obtained daily, and at d 0, 3, 5, 7, 10, and 14 post-infection (PI), 6 birds were euthanized, and a portion of the jejunum was removed for qRT-PCR. Infected birds had significantly decreased feed consumption between d 6 to 9 PI. At d 7 PI infected birds had a 45% reduction in weight gain, and pair-fed birds had a 32% reduction in weight gain. The feed conversion ratio at d 7 PI of infected birds was 2.2 while that of pair-fed birds was 1.7, compared to 1.5 in uninfected birds. Growth parameters were more affected in infected birds than in pair-fed birds. By measuring expression levels of nutrient uptake and processing genes via qRT-PCR, it was determined that genes encoding proteins located at the brush border of the gut epithelium were affected by infection as well as change in feed intake. The expression of AATs B°AT, b°,+AT, EAAT3, and PepT1 in infected birds decreased sharply at the height of infection; however, in birds that were pair fed, an increase in expression of b°,+AT, and PepT1 was observed, and little change was seen in expression of B°AT and EAAT3. In summary, the changes in expression of digestive enzymes and nutrient transporters are distinct between coccidia-infected birds compared to healthy pair-fed birds.

Differences in fecundity of Eimeria maxima strains exhibiting different levels of pathogenicity in its avian host.

Eimeria maxima is one of the most pathogenic species of avian coccidia, yet it is unknown why different E. maxima strains differ in the pathogenic effects they cause in chickens. The purpose of this study was to determine if a more pathogenic E. maxima strain (APU1) was also more fecund than a less pathogenic E. maxima strain (APU2). At identical doses, E. maxima APU1 always produces greater intestinal lesions and lower weight gain compared to E. maxima APU2. Using a dose response study, median and mean intestinal lesion scores in E. maxima APU1-infected chickens were greater by a score of 1-1.5 compared to chickens infected with E. maxima APU2. Likewise, weight gain depression in E. maxima APU1-infected chickens was 20-25% greater (equivalent to 110-130g body weight) than in E. maxima APU2-infected chickens. In order to understand the underlying cause of these observed clinical effects, 120 broiler chicks (5 oocyst levels, 6 replicates/level) were inoculated with various doses of E. maxima APU1 or APU2 oocysts. The dynamics of oocyst shedding was investigated by collecting fecal material every 12h from 114 to 210h post-inoculation (p.i.) and every 24h thereafter from 210 to 306h, and then processed for measuring E. maxima oocyst output. Oocysts were first observed at 138h p.i., and time of peak oocyst production was nearly identical for both E. maxima APU1 and APU2 around 150-162h. Total oocyst production was 1.1-2.6 fold higher at all dose levels for E. maxima APU1 compared to E. maxima APU2, being significantly higher (P<0.05) at the log 1.5 dose level. Other groups of chickens were infected with higher doses of E. maxima APU1 or APU2 oocysts, and intestinal lesions were assessed by histology at 72, 96, 120, and 144h p.i. Although schizonts, gamonts, and oocysts were observed at expected time-points, no obvious differences were noted in lesions induced by the two E. maxima strains. This study showed that the greater fecundity of E. maxima APU1 compared to E. maxima APU2 explains in part the observed differences in pathogenicity of the two E. maxima strains, but that other factors may contribute to differences in observed clinical effects.

Functional characterization of the turkey macrophage migration inhibitory factor.

Macrophage migration inhibitory factor (MIF) is a soluble protein that inhibits the random migration of macrophages and plays a pivotal immunoregulatory function in innate and adaptive immunity. The aim of this study was to clone the turkey MIF (TkMIF) gene, express the active protein, and characterize its basic function. The full-length TkMIF gene was amplified from total RNA extracted from turkey spleen, followed by cloning into a prokaryotic (pET11a) expression vector. Sequence analysis revealed that TkMIF consists of 115 amino acids with 12.5 kDa molecular weight. Multiple sequence alignment revealed 100%, 65%, 95% and 92% identity with chicken, duck, eagle and zebra finch MIFs, respectively. Recombinant TkMIF (rTkMIF) was expressed in Escherichia coli and purified through HPLC and endotoxin removal. SDS-PAGE analysis revealed an approximately 13.5 kDa of rTkMIF monomer containing T7 tag in soluble form. Western blot analysis showed that anti-chicken MIF (ChMIF) polyclonal antisera detected a monomer form of TkMIF at approximately 13.5 kDa size. Further functional analysis revealed that rTkMIF inhibits migration of both mononuclear cells and splenocytes in a dose-dependent manner, but was abolished by the addition of anti-ChMIF polyclonal antisera. qRT-PCR analysis revealed elevated transcripts of pro-inflammatory cytokines by rTkMIF in LPS-stimulated monocytes. rTkMIF also led to increased levels of IFN-γ and IL-17F transcripts in Con A-activated splenocytes, while IL-10 and IL-13 transcripts were decreased. Overall, the sequences of both the turkey and chicken MIF have high similarity and comparable biological functions with respect to migration inhibitory activities of macrophages and enhancement of pro-inflammatory cytokine expression, suggesting that turkey and chicken MIFs would be biologically cross-reactive.

Protection of Broiler Chicks Housed with Immunized Cohorts Against Infection with Eimeria maxima and E. acervulina.

The use of live oocyst vaccines is becoming increasingly important in the control of avian coccidiosis in broilers. Knowledge of the mechanisms employed when chicks uptake oocysts and become immune is important for optimizing delivery of live vaccines. The current study tests the hypothesis that chicks not initially immunized may ingest oocysts by contact with litter containing oocysts shed by immunized cohorts. In Experiment 1, day-old broiler chicks were housed in pens containing clean litter. In Trial 1, 100% of chicks in some pens were immunized with 2.5 X 10(3) Eimeria acervulina oocysts while in other pens only 75% of chicks were immunized and remaining cohorts within the pens were not immunized. Other pens contained chicks that served as nonimmunized nonchallenged controls or nonimmunized challenged controls (NIC). On day 21, birds were given a homologous challenge of 6 X 10(5) oocysts. A second identical trial was conducted, except birds were immunized with 500 Eimeria maxima oocysts and were challenged with 3 X 10(3) E. maxima oocysts. In Experiment 2, 100% of chicks in some pens were immunized with 500 E. acervulina oocysts while in other pens either 75% or 50% of the birds were immunized. On day 14, birds were challenged with 1 X 10(6) oocysts. Trial 2 was identical to Trial 1 except that birds were immunized with 100 E. maxima oocysts and challenged with 1 X 10(6) oocysts. For all experiments weight gain, feed conversion ratio (FCR), plasma carotenoids, and litter oocyst counts were measured. In Experiment 1, the level of protection in groups containing 25% nonimmunized cohorts, as measured by weight gain, carotenoid level, FCR, and oocyst litter counts, was identical to groups containing 100% immunized chicks. In Experiment 2, pens where 50% or 75% of birds were immunized with either E. maxima or E. acervulina were not well protected from decreases in weight gain and plasma carotenoids nor from increases in litter oocyst counts following a challenge infection administered on day 14 relative to NIC. In addition, pens of birds where 100% of chicks were immunized were not well protected compared to NIC, and resistance to coccidiosis infection in immunized chicks was less than resistance in chicks challenged at 21 days. These results in total suggest that, when birds are challenged after 21 days, cohorts are protected from detrimental effects of challenge infection. However, when challenge infection is given at 14 days, cohorts are not well protected. The results support a conclusion that protection to coccidiosis is conveyed to cohorts by contact with oocysts shed into the litter by immunized chicks, but this resistance may take 14 days to develop.

mRNA expression of amino acid transporters, aminopeptidase, and the di- and tri-peptide transporter PepT1 in the intestine and liver of posthatch broiler chicks.

Amino acid (AA) transporter proteins are responsible for the movement of amino acids in and out of cells. Aminopeptidase cleaves AAs from the N-terminus of polypeptides making them available for transport, while PepT1 is a di- and tripeptide transporter. In the intestine, these proteins are present on the brush border and basolateral membranes of enterocytes, and are essential for the uptake of AAs into enterocytes and their release into circulation. The purpose of this study was to determine the level of transcription of these genes after hatch in 3 regions of the small intestine, the ceca, and liver. Heritage broiler chicks (n=5) were sampled at day after hatch and days 3, 5, 7, 10, 12, 14, 17, and 21 posthatch, and mRNA expression level was measured using absolute quantitation. The small intestine (duodenum, jejunum, and ileum) expressed the largest quantities of each gene tested. The expression in the ceca and liver was 1 to 3 orders of magnitude less than that of the small intestine. The expression of basolateral transporters in the small intestine was more constant over days posthatch than the expression of brush border transporters. In the ceca the expression of the brush border transporters decreased over the sampling period, while expression of basolateral genes was relatively constant. In the liver the expression of Na+ independent cationic and zwitterionic amino acid transporter (bo,+AT), Na+ independent cationic amino acid transporter 2 (CAT2), excitatory amino acid transporter 3 (EAAT3), and the heavy chain corresponding to the bo,+) system (rBAT) significantly decreased at 12 days posthatch; however, the expression of Na+ independent cationic and Na+ dependent neutral amino acid transporter 1 (y+LAT1), Na+ coupled neutral amino acid transporter 1; (SNAT1), and Na+ coupled neutral amino acid transporter 2 (SNAT2) significantly increased at day 5 posthatch compared to day 1 and these levels remained throughout the rest of the sampling period. The current results suggest that at 1 day posthatch chicks are capable of AA processing and transport in the intestine as well as the liver. Additionally the ability of the ceca in transporting AA from the lumen may decrease with age. The liver should be capable of amino acid transport, but its capabilities may be more specific since the expression of several transporters in this organ is either absent or very low.

Evaluation of an experimental irradiated oocyst vaccine to protect broiler chicks against avian coccidiosis.

The current study investigates the use of irradiated oocysts to protect broiler chicks, raised on litter, from infection with multiple species of Eimeria. In order to determine the optimum radiation dose for each Eimeria species, 1-day-old chicks were immunized with oocysts of Eimeria maxima, Eimeria acervulina, or Eimeria tenella exposed to gamma radiation ranging from 0-500 Gy. The litter oocyst counts at 7 days postimmunization, and the effect on weight gain following a challenge infection, decreased with an optimum dose between 150-200 Gy. Based on this finding, broiler chicks were immunized with a mixture of E. maxima, E. acervulina, and E tenella that had been exposed to 150 or 200 Gy. This resulted in more than a 100-fold reduction in litter oocyst counts and significant protection from a challenge infection, as measured by improved weight gain and feed conversion ratio (FCR). Immunization of birds with oocysts receiving 200 Gy was less effective in providing protection from a challenge infection. An additional formulation of vaccines containing two different oocyst doses of the three species that had been irradiated with 150 Gy were evaluated in their ability to attenuate oocyst output and convey protection to challenge. Results were similar with both high and low numbers of irradiated oocysts. Immunized chicks shed less oocysts at 7 days postimmunization and were protected from negative effects of challenge infection as measured by FCR, changes in weight gain, lesion scores, and measurement of body composition. However, the level of protection was somewhat less than that achieved by immunization with nonirradiated oocysts. The overall conclusion is that an irradiated oocyst vaccine developed in this study can effectively protect chicks that are raised on litter from challenge infection with multiple species of Eimeria, comparable to vaccines with virulent or precocious strains.

Expression of nutrient transporters in duodenum, jejunum, and ileum of Eimeria maxima-infected broiler chickens.

The uptake of amino acids is mediated by active transporters located on the basolateral and brush border membranes of intestinal epithelial cells. The current study investigated the expression of amino acid transporters (AAT) and other genes in the intestine of chicks infected with Eimeria maxima. At 7-day postinfection (PI), tissue from each intestinal segment (duodenum, jejunum, and ileum) was taken from birds inoculated with 3 × 10(3) oocysts/bird and processed to recover RNA. Analysis of gene expression was performed using real-time reverse transcription polymerase chain reaction (qRT-PCR). Results were given as relative expression using ß2-microglobulin as an endogenous control. All the genes studied were expressed in three segments of the intestines, and expression of the genes was altered by infection with E. maxima. Even though the jejunum is considered the parasite's primary predilection site, there was no segment-related difference in expression of most of the genes studied. The antimicrobial peptide (LEAP2) was downregulated in all three segments of the intestine. The results also demonstrate that transporters associated with brush border membranes were downregulated while transporters associated with the basolateral membranes were upregulated and that E. maxima alters the expression of AAT and LEAP2 throughout the small intestine.

The mRNA expression of amino acid transporters, aminopeptidase N, and the di- and tri-peptide transporter PepT1 in the embryo of the domesticated chicken (Gallus gallus) shows developmental regulation.

The mRNA expression profile for 10 amino acid transporters, the di-and tri- peptide transporter (PepT1), and aminopeptidase N (APN) during chick embryogenesis was determined. Fertilized eggs were sampled at d 9, 11, 15, 17, 19, and 20 of incubation. Three to 4 embryos were sampled at each time period. At d 9 and 11, the entire intestine was collected due to its undifferentiated appearance. The ceca, duodenum, midgut, and liver were sampled at d 15, 17, 19, and 20. Gene expression was measured using absolute quantitation quantitative reverse-transcription PCR. In the liver, all genes except for PepT1 were expressed at most time points. At d 9, only the expression of Na⁺-independent cationic amino acid transporter 1, Na⁺-independent cationic amino acid transporter 2, and excitatory amino acid transporter 3 was detectable in the intestine, but by d 11, all genes associated with transporters of the basolateral surface were expressed, and at higher levels than genes associated with brush border transporters. By d 15, all of the genes tested were expressed in the duodenum, midgut, and ceca at high levels that remained relatively constant until d 20. Statistical analysis shows that at d 15, 17, 19, and 20 there is a significant interaction between the 2 main effects (days of incubation and region of the gut); therefore, it is likely that gene expression in different regions of the gut is dependent on the age of the embryo. At d 9 and 11, the gut may not function in amino acid uptake from the lumen and possibly relies on other structures such as the yolk sac. As the gut matures and protein becomes available in the lumen, amino acid transporters become highly expressed in all parts of the intestine. The data suggest that by d 15 of embryo development the gut may be capable of amino acid absorption.

Ostertagia ostertagi macrophage migration inhibitory factor is present in all developmental stages and may cross-regulate host functions through interaction with the host receptor.

Macrophage migration inhibitory factor (MIF) of Ostertagia ostertagi, an abomasal parasite of cattle, was characterised in the present study. Phylogenetic analysis identified at least three O. ostertagi MIFs (Oos-MIFs), each encoded by a distinct transcript: Oos-MIF-1.1, Oos-MIF-1.2 and Oos-MIF-2. Oos-MIF-2 is only distantly related to Oos-MIF-1s, but has higher sequence similarity with the Caenorhabditis elegans MIF2. Oos-MIF-1.1 and Oos-MIF-1.2 are similar (93%) and thus collectively referred to as Oos-MIF-1 when characterised with immunoassays. Recombinant Oos-MIF-1.1 (rOos-MIF-1.1) is catalytically active as a tautomerase. A mutation (rOos-MIF-1.1P1G) or duplication of Pro1 residue (rOos-MIF-1.1P1+P) resulted in reduced oligomerisation and loss of tautomerase activity. The tautomerase activity of rOos-MIF-1.1 was only partially inhibited by ISO-1 but was abrogated by a rOos-MIF-1.1-specific antibody. Oos-MIF-1 was detected in all developmental stages of O. ostertagi, with higher levels in the adult stage; it was also detected in adult worm excretory/secretory product. Oos-MIF-1 was localised to the hypodermis/muscle, reproductive tract and intestine, but not to the cuticle. rOos-MIF-1.1, but not rOos-MIF-1.1P1G, was able to specifically bind to human CD74, a MIF cell surface receptor, with an affinity comparable with human MIF. Immunostaining indicated that macrophages were able to internalise rOos-MIF-1.1, further supporting receptor-mediated transportation. Herein we also show that rOos-MIF-1.1 inhibited migration of bovine macrophages and restored glucocorticoid-suppressed, lipopolysaccharide-induced TNF-α and IL-8 in human and/or bovine macrophages. Given its dual role in self-regulation and molecular mimicry, this secreted parasite protein warrants investigation as a vaccine candidate against O. ostertagi infections in cattle.

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.

Characterization and localization of an Eimeria-specific protein in Eimeria maxima.

A recently completed analysis of Eimeria maxima transcriptome identified a gene with homology to sequences expressed by E. tenella and E. acervulina but lacking homology with other organisms including other apicomplexans. This gene, designated Eimeria-specific protein (ESP), codes for a protein with a predicted molecular weight of 19 kDa. The ESP gene was cloned and the recombinant protein expressed in bacteria and purified for preparation of specific antisera. Quantitative RT-PCR showed transcription of ESP was low in unsporulated oocysts and after 24 h of sporulation. However, transcription nearly doubled after 48 h of sporulation and reached its highest levels in sporozoites (SZ) and merozoites (MZ). The protein was detectable by Western blot in both sporulated oocysts and in SZ and MZ. Immuno-localization by light microscopy identified ESP in paired structures in the anterior of SZ and MZ. Immuno-localization by electron microscopy identified ESP in MZ rhoptries but no specific staining of any SZ structures was detected. In addition, localization studies on intestinal sections recovered from birds 120-h post-infection indicates that oocysts do not stain with anti-ESP but staining of microgametocytes and developing oocysts was observed. The results indicate that ESP is associated with the rhoptry of E. maxima and that the protein may have functions in other developmental stages.

Characterization of Neospora caninum macrophage migration inhibitory factor.

The present study is the first characterization of Neospora caninum macrophage migration inhibitory factor (NcMIF). BLAST-N analysis of NcMIF revealed high similarity (87%) to the Toxoplasma gondii MIF. NcMIF was cloned and expressed in Escherichia coli in 3 forms, NcMIF (mature protein), NcMIFm (mutation of proline-2 to glycine), and NcMIFhis (addition of a polyhistidine tag at the N-terminus). None of these recombinant NcMIFs (rNcMIF) had tautomerase, oxidoreductase, or immunologic regulatory activities. rNcMIF was unable to compete with recombinant human MIF for a MIF receptor (CD74), suggesting that NcMIF does not bind to this MIF receptor. The glycine substitution for proline-2 of NcMIF resulted in increased retention time on SEC-HPLC and decreased formation of dimers and trimers. The addition of N-terminal HIS-tag led to increased formation of trimers. Immunofluorescence staining demonstrated that NcMIF was localized to the apical end of N. caninum tachyzoites. Immunoelectron microscopy further revealed that NcMIF was present in the micronemes, rhoptries, dense granules, and nuclei. NcMIF was abundant in the tachyzoite lysate and present in excretory and secretory antigen (ESAg) preparations. Total and secretory NcMIF was more abundant in a non-pathologic clone, Ncts-8, than in the wild type isolate (NC1). Furthermore, NcMIF release by the both isolates was increased in the presence of calcium ionophore. This differential production of NcMIF by the pathologic and non-pathologic isolates of N. caninum may suggest a critical role of this molecule in the infectious pathogenesis of this parasite.

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.

Macrophage migration inhibitory factor (MIF) of the protozoan parasite Eimeria influences the components of the immune system of its host, the chicken.

Macrophage migration inhibitory factor (MIF) is a soluble factor produced by sensitized T lymphocytes that inhibits the random migration of macrophages. Homologues of MIF from invertebrates have been identified, making it an interesting molecule from a functional perspective. In the present study, the localization of a parasite MIF protein as well as its effect on the host was characterized. Western blot analysis shows that Eimeria MIF (EMIF) is found during all parasite developmental stages tested. Transmission electron microscopy shows that MIF is distributed throughout cytosol and nucleus of Eimeria acervulina merozoites. Immunohistochemical analysis suggests that EMIF may be released into the surrounding tissues as early as 24 h after infection, while later during oocyst formation, MIF expression is localized to areas immediately surrounding the oocysts, as well as in wall-forming bodies. The chemotaxis assay revealed an inhibitory function of EMIF on chicken monocyte migration. Quantitative real-time PCR was performed to examine the effect of EMIF on host immune system by measuring the transcripts of inflammatory mediators. An ex vivo stimulation study showed that E. acervulina MIF (EaMIF) enhanced expression of pro-inflammatory cytokines and chemokines in the presence of lipopolysaccharide (LPS). Furthermore, sequential treatment of adherent peripheral blood mononuclear cells with EaMIF, chicken MIF, and LPS in 2-h intervals led to the highest levels of interleukin (IL)-1B, chemokine CCLi3, IL-18, and interferon-gamma mRNA expression. This study shows that parasite MIF is widely expressed and may have potential effects on the immune system of the host.

The use of dual-energy X-ray absorptiometry to assess the impact of Eimeria infections in broiler chicks.

A number of parameters have been used to assess the impact ofcoccidiosis on chickens in clinical settings as well as in experimental studies. However, a rapid way to determine body composition would be useful to evaluate or compare responses to coccidia and could give further insight into the metabolic impact of infection. The current study evaluates the use of dual X-ray absorptiometry (DEXA) to determine the impact of coccidiosis on body composition in chicks receiving inoculations with single or mixed species of Eimeria. Chicks infected with Eimeria maxima, Eimeria acervulina, or Eimeria tenella had altered parameters of body composition as measured by DEXA at 6 days postinfection (PI). The greatest effects were noted in birds infected with E. acervulina or E. maxima, where lean mass and fat were reduced from control values about 75% and 85%, respectively. In chicks infected with E. tenella, tissue and fat were reduced about 10%. Bone mineral content (BMC) was about 75% of control values in birds infected with E. acervulina or E. maxima, but only E. acervulina altered bone mineral density (BMD). The decreases in BMC and BMD are likely due to malabsorption. In chicks receiving a mixed coccidian infection, all DEXA parameters were significantly decreased at 8 days PI compared with age-matched controls. As with single infections, BMD and BMC were significantly depressed (P < 0.05). Values of all DEXA parameters were near 92% of control values by day 16 PI. Analysis of all birds in the current study indicates DEXA tissue weight slightly underestimated the gravimetrically measured weight by about 3%. The current results demonstrate that DEXA is a potentially important tool for the rapid evaluation of the effect of coccidiosis on broiler chicks and suggest it can be useful for evaluation of vaccines and other disease controls.

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.