Search

Coccidiosis is one of the most prevalent diseases seen in the poultry industry leading to excessive economic losses. The aim of this study was to investigate the effect of butyric acid glycerol esters (BE) on the ileal and cecal microbiota in birds challenged with Eimeria maxima (EM). Ross 708 male broilers were fed a diet supplemented with 0 (control) or 0.25% BE from day 1. On day 21, half of the birds were infected with 103 EM oocysts. For determing microbiota, ileal and cecal contents and epithelial scrapings were collected at 7 and 10 D postinfection (PI). Alpha diversity of bacterial communities was mostly affected (P < 0.05) by time PI and EM infection. The richness of luminal bacterial populations in the ileum and ceca was affected (P < 0.05) by addition of BE and by time PI × EM × BE interaction, respectively. In the ileal and cecal luminal and mucosal bacterial communities, permutational multivariate analysis of variance (PERMANOVA, unweighted UniFrac) showed significant (P < 0.05) differences because of time PI and interaction between time PI, EM, and BE. Significant (P < 0.05) differences in taxonomic composition at the family level were observed in microbiota of luminal and mucosal populations of the ileum and ceca owing to time PI, EM, BE, and their interactions. The bacterial community present in the cecal lumen was characterized by the lowest number of differential bacteria, whereas the cecal mucosal community was characterized by the highest number of differentially abundant bacteria. In conclusion, our results show that EM infection and time PI has the biggest impact on microbial diversity in the chicken gut. The presence of BE in the diet had a limited effect on gut microbiota.

Eimeria parasites cause enteric disease in livestock and the closely related Cyclospora cayetanensis causes human disease. Oocysts of these coccidian parasites undergo maturation (sporulation) before becoming infectious. Here, we assessed transcription in maturing oocysts of Eimeria acervulina, a widespread chicken parasite, predicted gene functions, and determined which of these genes also occur in C. cayetanensis. RNA-Sequencing yielded ~2 billion paired-end reads, 92% of which mapped to the E. acervulina genome. The ~6,900 annotated genes underwent temporally-coordinated patterns of gene expression. Fifty-three genes each contributed >1,000 transcripts per million (TPM) throughout the study interval, including cation-transporting ATPases, an oocyst wall protein, a palmitoyltransferase, membrane proteins, and hypothetical proteins. These genes were enriched for 285 gene ontology (GO) terms and 13 genes were ascribed to 17 KEGG pathways, defining housekeeping processes and functions important throughout sporulation. Expression differed in mature and immature oocysts for 40% (2,928) of all genes; of these, nearly two-thirds (1,843) increased their expression over time. Eight genes expressed most in immature oocysts, encoding proteins promoting oocyst maturation and development, were assigned to 37 GO terms and 5 KEGG pathways. Fifty-six genes underwent significant upregulation in mature oocysts, each contributing at least 1,000 TPM. Of these, 40 were annotated by 215 GO assignments and 9 were associated with 18 KEGG pathways, encoding products involved in respiration, carbon fixation, energy utilization, invasion, motility, and stress and detoxification responses. Sporulation orchestrates coordinated changes in the expression of many genes, most especially those governing metabolic activity. Establishing the long-term fate of these transcripts in sporulated oocysts and in senescent and deceased oocysts will further elucidate the biology of coccidian development, and may provide tools to assay infectiousness of parasite cohorts. Moreover, because many of these genes have homologues in C. cayetanensis, they may prove useful as biomarkers for risk.

Butyrate is a feed additive that has been shown to have antibacterial properties and improve gut health in broilers. Here, we examined the performance and gene expression changes in the ileum of tributyrin–supplemented broilers infected with coccidia. Ninety-six, Ross 708 broilers were fed either a control corn–soybean–based diet (−BE) or a diet supplemented with 0.25% (w/w) tributyrin (+BE). Birds were further divided into groups that were inoculated with Eimeria maxima oocysts (EM) or sham-inoculated (C) on day 21 posthatch. At 7 d postinfection (7 d PI), the peak of pathology in E. maxima infection, tributyrin-supplemented birds had significantly improved feed conversion ratios (FCR, P < 0.05) and body weight gain (BWG, P < 0.05) compared with -BE-infected birds, despite both groups having similar feed intake (FI, P > 0.05). However, at 10 d post-infection (10 d PI) no significant effects of feed type or infection were observed. Gene expression in the ileum was examined for insights into possible effects of infection and tributyrin supplementation on genes encoding proteins related to immunity, digestion, and gut barrier integrity. Among immune-related genes examined, IL-1B and LEAP2 were only significantly affected at 7 d PI. Transcription of genes related to digestion (APN, MCT1, FABP2, and MUC2) were primarily influenced by infection at 7 d PI and tributyrin supplementation (FABP2 and MUC2) at 10 d PI. With exception of ZO1, tight junction genes were affected by either infection or feed type at 7 d PI. At 10 d PI, only CLDN1 was not affected by either infection or feed type. Overall tributyrin shows promise as a supplement to improve performance during coccidiosis in broiler chickens; however, its effect on gene expression and mode of action requires further research.

A programmed self-destructive Salmonella vaccine delivery system was developed to facilitate efficient colonization in host tissues that allows release of the bacterial cell contents after lysis to stimulate mucosal, systemic, and cellular immunities against a diversity of pathogens. Adoption and modification of these technological improvements could form part of an integrated strategy for cost-effective control and prevention of infectious diseases, including those caused by parasitic pathogens. Avian coccidiosis is a common poultry disease caused by Eimeria. Coccidiosis has been controlled by medicating feed with anticoccidial drugs or administering vaccines containing low doses of virulent or attenuated Eimeria oocysts. Problems of drug resistance and nonuniform administration of these Eimeria resulting in variable immunity are prompting efforts to develop recombinant Eimeria vaccines. In this study, we designed, constructed, and evaluated a self-destructing recombinant attenuated Salmonella vaccine (RASV) lysis strain synthesizing the Eimeria tenella SO7 antigen. We showed that the RASV lysis strain χ11791(pYA5293) with a ΔsifA mutation enabling escape from the Salmonella-containing vesicle (or endosome) successfully colonized chicken lymphoid tissues and induced strong mucosal and cell-mediated immunities, which are critically important for protection against Eimeria challenge. The results from animal clinical trials show that this vaccine strain significantly increased food conversion efficiency and protection against weight gain depression after challenge with 105E. tenella oocysts with concomitant decreased oocyst output. More importantly, the programmed regulated lysis feature designed into this RASV strain promotes bacterial self-clearance from the host, lessening persistence of vaccine strains in vivo and survival if excreted, which is a critically important advantage in a vaccine for livestock animals. Our approach should provide a safe, cost-effective, and efficacious vaccine to control coccidiosis upon addition of additional protective Eimeria antigens. These improved RASVs can also be modified for use to control other parasitic diseases infecting other animal species.

Fayoumi chickens are believed to be more disease resistant compared to commercial broiler chickens. The objective of this study was to compare mRNA expression of intestinal nutrient transporters, digestive enzymes, and host defense peptides (HDP) between Eimeria maxima-challenged Fayoumi and Ross broiler chickens. At 21 d of age, Ross broilers and Fayoumi lines M5.1 and M15.2 were challenged with 1,000 E. maxima oocysts. Control birds were not challenged. Duodenum, jejunum, and ileum were sampled (n = 6) at 7 d post challenge. Gene expression was analyzed using relative quantification PCR. Data were analyzed by ANOVA and significance level was set at P< 0.05. There was numerical, but not statistically significant, differential weight gain depression for Ross (15%) and Fayoumi lines M5.1 (21%) and M15.2 (22%) and significant line-specific changes in gene expression. For nutrient transporters, there was downregulation of mRNA for the brush border membrane, amino acid transporters b(0,+) AT/rBAT, BoAT, and EAAT3 in different segments of the small intes-tine of Ross and both lines of Fayoumi chickens, indicating that E. maxima challenge likely caused a decrease in nutrient uptake. For HDP, there was downregulation of avian beta defensin (AvBD) 1, 6, 10, 12, and 13 mRNA in the jejunum of the 2 Fayoumi lines, but no change in the Ross broilers. In the duodenum, there was upregulation of AvBD10 mRNA in the Ross and both Fayoumi lines and additionally upregulation of AvBD11, 12, and 13 mRNA in only Fayoumi line M15.2. Liver expressed antimicrobial peptide 2 (LEAP2) mRNA was downregulated in the duodenum and jejunum of Ross and Fayoumi line M5.1 but not in Fayoumi line M15.2. The homeostatic, non-challenged levels of AvBD mRNA were greater in Fayoumi line M15.2 than Ross and Fayoumi line M5.1 in the duodenum and ileum. This study demonstrates tissueand genetic line-specific transcriptional responses to E. maxima, highlights novel potential candidate genes for response to coccidiosis, and confirms a role for several previously reported genes in response to coccidiosis. College of Agriculture and Life Sciences Pratt Endowment at Virginia Tech; Virginia Agricultural Experiment Station; Hatch Program of the National Institute of Food and Agriculture, U.S. Department of Agriculture; Animal Health Formula Funds; Hatch project, Iowa State University [5458] This work was funded in part by the College of Agriculture and Life Sciences Pratt Endowment at Virginia Tech (SS), the Virginia Agricultural Experiment Station and the Hatch Program of the National Institute of Food and Agriculture, U.S. Department of Agriculture (EAW), and Animal Health Formula Funds and Hatch project number 5458, Iowa State University (SJL). Public domain – authored by a U.S. government employee

Ostertagiosis remains an economically important parasitic disease in cattle in the temperate regions of the world. Repeated exposures to Ostertagia ostertagi in calves cause significant pathology in the abomasum but elicit little protective immunity. The larvae use the host’s gastric glands as a niche for development, where the parasite completes its parasitic stages, while in the gastric glands, the larvae must down-regulate the host inflammatory immune responses. Annexin (ANX) A1, commonly found in most eukaryotes, is heavily involved in controlling anti-inflammatory responses by binding receptors on leukocytes. We hypothesized, therefore, that parasite proteins of the ANX family may be involved in host-parasite interactions during ostertagiosis. BLASTN search with the bovine ANXA1 identified two families of Oos-ANX like proteins (Oos-ANXL), each of which was highly conserved at the genetic level and identical at the amino acid sequence level. Oos-ANXL-1 is encoded by two transcripts and Oos-ANXL-2 by 20 transcripts. The present study characterized one Oos-ANXL, representing the most abundant Oos-ANXL, which was further defined as Oost-ANXL-2.1. Oos-ANXL-2.1 with a coding sequence of 519 bp was PCR-amplified, cloned, and expressed. Oos-ANXL-2.1 was immunolocalized to both L3 and adult, but not L4. The staining appeared to be associated with the gut and hypodermis in L3, but it was specifically localized to the hypodermis in adult worms. Western blots detected three protein bands in parasite lysates using anti-recombinant Oos-ANXL-2.1 antibody. Integrated optical density for each of the 3 Oos-ANXL-2s or the total Oos-ANXL-2s detected by Western blots (P

The aim of the present study was to evaluate oocyst shedding in cats immunized by nasal route with T. gondii proteins ROP2. Twelve short hair cats (Felis catus) were divided in three groups G1, G2 and G3 (n=4). Animals from G1 received 100 μg of rROP2 proteins plus 20 μg of Quil-A, G2 received 100 μg of BSA plus 20 μg of Quil-A, and the G3 only saline solution (control group). All treatments were done by intranasal route at days 0, 21, 42, and 63. The challenge was performed in all groups on day 70 with ≅ 800 tissue cysts of ME-49 strain by oral route. Animals from G1 shed less oocysts (86.7%) than control groups. ELISA was used to detect anti-rROP2 IgG and IgA, however, there were no correlation between number of oocyst shedding by either IgG or IgA antibody levels. In the present work, in spite of lesser oocysts production in immunized group than control groups, it was not possible to associate the use of rROP2 via nostrils with protection against oocyst shedding. For the future, the use of either other recombinant proteins or DNA vaccine, in combination with rROP2 could be tested to try improving the efficacy of this kind of vaccine.

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 (P0.05) 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.

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.

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).

Eimeria, protozoan parasites from the phylum Apicomplexa, can cause the enteric disease coccidiosis in all farmed animals. Coccidiosis is commonly considered to be most significant in poultry; due in part to the vast number of chickens produced in the World each year, their short generation time, and the narrow profit margins associated with their production. Control of Eimeria has long been dominated by routine chemoprophylaxis, but has been supplemented or replaced by live parasite vaccination in a minority of production sectors. However, public and legislative demands for reduced drug use in food production is now driving dramatic change, replacing reliance on relatively indiscriminate anticoccidial drugs with vaccines that are Eimeria species-, and in some examples, strain-specific. Unfortunately, the consequences of deleterious selection on Eimeria population structure and genome evolution incurred by exposure to anticoccidial drugs or vaccines are unclear. Genome sequence assemblies were published in 2014 for all seven Eimeria species that infect chickens, stimulating the first population genetics studies for these economically important parasites. Here, we review current knowledge of eimerian genomes and highlight challenges posed by the discovery of new, genetically cryptic Eimeria operational taxonomic units (OTUs) circulating in chicken populations. As sequencing technologies evolve understanding of eimerian genomes will improve, with notable utility for studies of Eimeria biology, diversity and opportunities for control.

Avian coccidiosis is a disease caused by the intestinal protozoa Eimeria. The site of invasion and lesions in the intestine is species-specific, for example E. acervulina affects the duodenum, E. maxima the jejunum, and E. tenella the ceca. Lesions in the intestinal mucosa cause reduced feed efficiency and body weight gain. The growth reduction may be due to changes in expression of digestive enzymes and nutrient transporters in the intestine. The objective of this study was to compare the expression of digestive enzymes, nutrient transporters and an antimicrobial peptide in broilers challenged with either E. acervulina, E. maxima or E. tenella. The genes examined included digestive enzymes (APN and SI), peptide and amino acid transporters (PepT1, ASCT1, b(0,+)AT/rBAT, B(0)AT, CAT1, CAT2, EAAT3, LAT1, y(+)LAT1 and y(+)LAT2), sugar transporters (GLUT1, GLUT2, GLUT5 and SGLT1), zinc transporter (ZnT1) and an antimicrobial peptide (LEAP2). Duodenum, jejunum, ileum and ceca were collected 7 days post challenge. E. acervulina challenge resulted in downregulation of various nutrient transporters or LEAP2 in the duodenum and ceca, but not the jejunum or ileum. E. maxima challenge produced both downregulation and upregulation of nutrient transporters and LEAP2 in all three segments of the small intestine and ceca. E. tenella challenge resulted in the downregulation and upregulation of nutrient transporters and LEAP2 in the jejunum, ileum and ceca, but not the duodenum. At the respective target tissue, E. acervulina, E. maxima and E. tenella infection caused common downregulation of APN, b(0,+)AT, rBAT, EAAT3, SI, GLUT2, GLUT5, ZnT1 and LEAP2. The downregulation of nutrient transporters would result in a decrease in the efficiency of protein and polysaccharide digestion and uptake, which may partially explain the weight loss. The downregulation of nutrient transporters may also be a cellular response to reduced expression of the host defense protein LEAP2, which would diminish intracellular pools of nutrients and inhibit pathogen replication.

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.

Previous studies comparing the genome sequences of Cryptosporidium parvum with Cryptosporidium hominis identified a number of highly divergent genes that might reflect positive selection for host specificity. In the present study, the C. parvum DNA sequence cgd8-5370, which encodes a protein whose amino acid sequence differs appreciably from its homologue in C. hominis , was cloned by PCR and expressed as a recombinant protein in Escherichia coli . Antisera raised against the recombinant cgd8-5370 antigen strongly recognized a unique 33 kDa protein in immunoblots from reducing and non-reducing SDS-PAGE of native C. parvum protein. However, anti-Cp33 sera did not recognize the native 33 kDa homologue in C. hominis . In an immunofluorescence assay (IFA), anti-Cp33 serum recognized an antigen in the anterior end of air-dried C. parvum sporozoites but failed to bind at any sites in C. hominis sporozoites, indicating its specificity for C. parvum . IFA staining of live C. parvum sporozoites with anti-Cp33 serum failed to bind to the parasite, indicating that the CP33 antigen is not on the sporozoite surface, which is consistent with topology predictions based on the encoded amino acid sequence. RT-PCR analysis of cgd8-5370 mRNA before or during C. parvum oocyst excystation revealed transcripts only in excysting sporozoites. Thus, Cp33 represents one of a small number of proteins shown to differentiate C. parvum from C. hominis sporozoites and oocysts.

Neospora caninum is a common cause of abortion in cattle worldwide. Canids, including the dog and the dingo (Canis familiaris), the coyote (Canis latrans), and the gray wolf (Canis lupus) are its definitive hosts that can excrete environmentally resistant oocysts in the environment, but also can act as intermediate hosts, harboring tissue stages of the parasite. In an attempt to isolate viable N. caninum from tissues of naturally infected wolves, brain and heart tissue from 109 wolves from Minnesota were bioassayed in mice. Viable N. caninum (NcWolfMn1, NcWolfMn2) was isolated from the brains of two wolves by bioassays in interferon gamma gene knockout mice. DNA obtained from culture-derived N. caninum tachyzoites of the two isolates were analyzed by N. caninum-specific Nc5 polymerase chain reaction and confirmed diagnosis. This is the first report of isolation of N. caninum from tissues of any wild canid host.

Coccidiosis is a major contributor to economic losses in the poultry industry due to its detrimental effects on growth performance and nutrient utilization. We hypothesized that the combined effects of supplemental dietary Thr and purified fiber may modulate the intestinal environment and positively affect intestinal immune responses and barrier function in broiler chicks infected with Eimeria maxima. A Thr-deficient basal diet (3.1 g of Thr/kg of diet) was supplemented with 70 g/kg of silica sand (control) or high-methoxy pectin and 1 of 2 concentrations of Thr (1.8 or 5.3 g/kg of diet; 4 diets total), and fed to chicks from hatch to d 16 posthatch. On d 10 posthatch, chicks received 0.5 mL of distilled water or an acute dose of Eimeria maxima (1.5 × 10(3) sporulated oocytes) with 6 replicate pens of 6 chicks per each of 8 treatment combinations (4 diets and 2 inoculation states). Body weight gain, feed intake, and G:F increased (P < 0.01) with addition of 5.3 g of Thr/kg of diet. Eimeria maxima schizonts were present only in intestinal tissue sampled from infected birds (P < 0.01). Weights of cecal digesta were highest (P < 0.01) in pectin-fed birds, and ceca with the heaviest weights also had the highest concentrations of total short-chain fatty acids. Expression of interleukin-12 in ileal mucosa was highest (P < 0.01) in infected birds receiving the control diet with 5.3 g of supplemental Thr/kg. In cecal tonsils, interferon-γ expression was highest in infected birds receiving the control diet (fiber × infection, P < 0.05); interferon-γ expression was lowest in infected birds fed the high Thr diet (Thr × infection, P < 0.05). There were no differences due to infection or Thr supplementation for cytokine expression in birds fed pectin-containing treatments. Overall, we conclude that although pectin has some protective function against coccidiosis, Thr supplementation had the greatest effect on intestinal immune response and maintenance of near normal growth in young broiler chicks infected with E. maxima.

Avian coccidiosis is caused by the intracellular protozoan Eimeria, which produces intestinal lesions leading to weight gain depression. Current control methods include vaccination and anticoccidial drugs. An alternative approach involves modulating the immune system. The objective of this study was to profile the expression of host defense peptides such as avian beta-defensins (AvBDs) and liver expressed antimicrobial peptide 2 (LEAP2), which are part of the innate immune system. The mRNA expression of AvBD family members 1, 6, 8, 10, 11, 12, and 13 and LEAP2 was examined in chickens challenged with either E. acervulina, E. maxima, or E. tenella. The duodenum, jejunum, ileum, and ceca were collected 7 d post challenge. In study 1, E. acervulina challenge resulted in down-regulation of AvBD1, AvBD6, AvBD10, AvBD11, AvBD12, and AvBD13 in the duodenum. E. maxima challenge caused down-regulation of AvBD6, AvBD10, and AvBD11 in the duodenum, down-regulation of AvBD10 in the jejunum, but up-regulation of AvBD8 and AvBD13 in the ceca. E. tenella challenge showed no change in AvBD expression in any tissue. In study 2, which involved challenge with only E. maxima, there was down-regulation of AvBD1 in the ileum, AvBD11 in the jejunum and ileum, and LEAP2 in all 3 segments of the small intestine. The expression of LEAP2 was further examined by in situ hybridization in the jejunum of chickens from study 2. LEAP2 mRNA was expressed similarly in the enterocytes lining the villi, but not in the crypts of control and Eimeria challenged chickens. The lengths of the villi in the Eimeria challenged chickens were less than those in the control chickens, which may in part account for the observed down-regulation of LEAP2 mRNA quantified by PCR. Overall, the AvBD response to Eimeria challenge was not consistent; whereas LEAP2 was consistently down-regulated, which suggests that LEAP2 plays an important role in modulating an Eimeria infection.

As a member of the interleukin (IL)-10 family, IL-22 is an important mediator in modulating tissue responses during inflammation. Through activation of STAT3-signaling cascades, IL-22 induces proliferative and anti-apoptotic pathways, as well as antimicrobial peptides (AMPs), that help prevent tissue damage and aid in its repair. This study reports the cloning and expression of recombinant chicken IL-22 (rChIL-22) and its soluble receptor, rChIL22BP, and characterization of biological effects of rChIL-22 during inflammatory responses. Similar to observations with mammalian IL-22, purified rChIL-22 had no effect on either peripheral blood mononuclear cells (PBMCs) or lymphocytes. This was due to the low expression of the receptor ChIL22RA1 chain compared to ChIL10RB chain. rChIL-22 alone did not affect chicken embryo kidney cells (CEKCs); however, co-stimulation of CEKCs with LPS and rChIL-22 enhanced the production of pro-inflammatory cytokines, chemokines and AMPs. Furthermore, rChIL-22 alone stimulated and induced acute phase reactants in chicken embryo liver cells (CELCs). These effects of rChIL-22 were abolished by pre-incubation of rChIL-22 with rChIL22BP. Together, this study indicates an important role of ChIL-22 on epithelial cells and hepatocytes during inflammation.

The present study analyzed giardin transcription in trophozoites and cysts during encystation of Giardia lamblia . Encystment was induced using standard methods, and the numbers of trophozoites and cysts were counted at various time points during the process. At all time points, RNA from both stages were assayed for levels of alpha2-, beta-, and delta-giardin mRNA as well as for cyst wall protein 3 (CWP3) mRNA using quantitative RT-PCR. In encystation medium, the number of G. lamblia trophozoites decreased, while the number of cysts increased between 0 and 72 hr. In trophozoites, alpha2- and beta-giardin transcription decreased over time, while delta-giardin transcription remained unchanged during the same time period. CWP3 transcription exhibited a slight increase in trophozoites at 8 hr, followed by a decrease at subsequent time points. Expression of alpha2-giardin increased at 48 hr in cysts followed by decreased expression at 72 hr, while beta- and delta-giardin expression was unchanged during encystation. CWP3 transcription gradually decreased from 24-72 hr in cysts. Consistent with previous studies, giardin proteins appeared to be disassembled into amorphous structures inside cysts during encystation. These findings represent the first analysis of giardin transcription in separate populations of trophozoites and cysts during encystation and indicate differential regulation of giardin mRNA expression by these developmental stages.

Macrophage migration inhibitory factor (MIF) is recognized as a soluble factor produced by sensitized T lymphocytes and inhibits the random migration of macrophages. Recent studies have revealed a more prominent role for MIF as a multi-functional cytokine mediating both innate and adaptive immune responses. This study describes the cloning and functional characterization of avian MIF in an effort to better understand its role in innate and adaptive immunity, and potential use in poultry health applications. The full-length avian MIF gene was amplified from stimulated chicken lymphocytes and cloned into a prokaryotic expression vector. The confirmed 115 amino acid sequence of avian MIF has 71% identity with human and murine MIF. The bacterially expressed avian recombinant MIF (rChMIF) was purified, followed by endotoxin removal, and then tested by chemotactic assay and quantitative real-time PCR (qRT-PCR). Diff-Quick staining revealed a substantial decrease in migration of macrophages in the presence of 0.01microg/ml rChMIF. qRT-PCR analysis revealed that the presence of rChMIF enhanced levels of IL-1beta and iNOS during PBMCs stimulation with LPS. Additionally, the Con A-stimulated lymphocytes showed enhanced interferon (IFN)-gamma and IL-2 transcripts in the presence of rChMIF. Interestingly, addition of rChMIF to the stimulated PBMCs, in the presence of lymphocytes, showed anti-inflammatory function of rChMIF. To our knowledge, this study represents the first report for the functional characterization of avian MIF, demonstrating the inhibition of macrophage migration, similar to mammalian MIF, and the mediation of inflammatory responses during antigenic stimulation.

Challenge infections with 10(3), 5 x 10(4), 10(5), or 5 x 10(5) sporulated Eimeria praecox oocysts caused moderate but significant weight gain reduction at all infective doses. Substantial reduction in plasma carotenoids and moderate but significant increases in plasma NO2(-) + NO3- were observed only at the two higher doses when measured at day 6 postchallenge (PC). Daily monitoring of chickens after challenge with 5 x 10(4) oocysts revealed an inflammatory response in the duodenum and jejunum beginning at day 1 PC that was associated with a significant increase in levels of plasma NO2(-) + NO3-, which peaked at day 4 PC. A moderate, uniform hyperplasia of the small intestine and significant depression of plasma carotenoids were observed on days 4-6 PC. Plasma NO2(-) + NO3- decreased to control levels by day 6 PC. All infections were accompanied by production of a mucoid exudate in the duodenum and jejunum, which became thick and opaque by 4 days PC and tended to obscure mildly inflamed areas. These observations indicate that the acute host response to primary infection with E. praecox is both different from and occurs earlier than the response to experimental infections with other Eimeria spp., such as Eimeria acervulina, Eimeria maxima, or Eimeria tenella. These factors need to be considered in observations of pathology arising from co-infections of E. praecox with other Eimeria species, especially in drug sensitivity testing of Eimeria oocysts recovered from litter and in the evaluation of live oocyst vaccines.

The impact of coccidiosis outbreaks on the productivity of broiler chicken farms can be substantial, depending on the severity of disease caused by particular species and strains of Eimeria. We examined the genetic diversity of Eimeria species present in commercial broiler farms in relation to their performance level. Four groups of broiler chicken farms in Arkansas (AR) and North Carolina (NC), having either high or low performance levels, were sampled for Eimeria spp. oocysts. We amplified gDNA from oocysts by using genus-specific primers targeting 18S ribosomal RNA, the first and second internal transcribed spacer regions, and cytochrome c oxidase subunit I as the established species-specific primers. Eimeria spp. diversity was not homogenous among the 4 farm groups, with less-pathogenic species (E. mitis and E. mivati-like) associated with AR and NC high-performance farms, respectively, and a pathogenic species (E. brunetti) associated with AR low-performance farms. Sequence analyses identified multiple E. maxima and E. mitis genetic variants, from which 2 E. maxima variants were unique to low-performance farms. Distinct populations of sequences at the NC high-performance farms were identified as E. mivati-like, based on homology searches. Our study demonstrated the utility of analyzing multiple genomic loci to assess composition and polymorphisms of Eimeria spp. populations.

A cDNA coding for detlaq-giardin was cloned from Giardia lamblia trophozoites to localize the protein and to study its function in mediating surface attachment. Recombinant delta-giardin antigen was expressed in Escherichia coli as a poly-histidine fusion protein and was purified by affinity chromatography for production of antisera to delta-giardin. By immunoblotting analysis, antisera to recombinant delta-giardin antigen recognized a 31-kDa protein on G. lamblia trophozoites. Anti-recombinant delta-giardin was used to localize the native protein to the trophozoite ventral disk in both immunofluorescence and immunoelectron microscopy assays. Pre-treatment of G. lamblia trophozoites with anti-delta-giardin sera caused morphological changes in the parasite and inhibited trophozoite binding to the surface of cell culture slides. Binding of antibodies to delta-giardin may provide a means of inhibiting attachment of G. lamblia trophozoites to the intestinal epithelium and thereby prevent clinical giardiasis.

Recombinant attenuated Salmonella vaccines against avian coccidiosis were developed to deliver Eimeria species antigens to the lymphoid tissues of chickens via the type 3 secretion system (T3SS) and the type 2 secretion system (T2SS) of Salmonella . For antigen delivery via the T3SS, the Eimeria tenella gene encoding sporozoite antigen SO7 was cloned downstream of the translocation domain of the Salmonella enterica serovar Typhimurium sopE gene in the parental pYA3868 and pYA3870 vectors to generate pYA4156 and pYA4157. Newly constructed T3SS vectors were introduced into host strain χ8879 (Δ phoP233 Δ sptP1033 :: xylE Δ asdA16 ), an attenuated derivative of the highly virulent UK-1 strain. The SopE-SO7 fusion protein was secreted by the T3SS of Salmonella . The vector pYA4184 was constructed for delivery of the SO7 antigen via the T2SS. The SO7 protein was toxic to Salmonella when larger amounts were synthesized; thus, the synthesis of this protein was placed under the control of the lacI repressor gene, whose expression in turn was dependent on the amount of available arabinose in the medium. The pYA4184 vector was introduced into host strain χ9242 (Δ phoP233 Δ asdA16 Δ araBAD23 Δ relA198 :: araC P BAD lacI TT [TT is the T4 ipIII transcription terminator]). In addition to SO7, for immunization and challenge studies we used the EAMZ250 antigen of Eimeria acervulina , which was previously shown to confer partial protection against E. acervulina challenge when it was delivered via the T3SS. Immunization of chickens with a combination of the SO7 and EAMZ250 antigens delivered via the T3SS induced superior protection against challenge by E. acervulina . In contrast, chickens immunized with SO7 that was delivered via the T2SS of Salmonella were better protected from challenge by E. tenella .

The effect of Eimeria praecox on concurrent Eimeria maxima infection was studied in susceptible chickens. Cinical signs of coccidiosis were assessed in single E. praecox or E. maxima infections and compared to dual infection with both Eimeria species. Groups infected solely with 10 4 E. maxima oocysts displayed weight gains that were 48% of weight gain in uninfected controls. Weight gain in chickens infected only with 10 4 E. praecoxoocysts was 90% of uninfected controls. Averageweight gain in chickens infected with both E. maxima and E. praecox was 79% of controls, and showed no significant difference (P > 0.05) from weight gaininE.praecox-infectedchickens.Feedutilization(feed conversionratio,FCR)inchickensinfectedwithbothspeciesshowedno significant difference (P > 0.05) from FCR in non-infected controls or chickens infected with E. praecox alone; all showing a significant difference (P < 0.05) from FCR in chickens infected solely with E. maxima. Although E. praecox did not appear to have a negative effect on weight gain and FCR, it did cause a significant decrease in serum carotenoids. Analysis of oocysts excreted by chickens during dual infection showed little effect of E. praecox on E. maxima oocyst production. Published by Elsevier B.V.

The developmental expression of the antigen SO7, which has been previously shown to protect chickens against infection by several Eimeria species, was investigated. Using RT-PCR, mRNA for SO7 was found to be restricted primarily to unsporulated oocysts (0 hr). Western blot (WB) analysis with an antibody to recombinant SO7 (rbSO7) revealed expression of the protein from 6 to 72 hr (fully sporulated) of sporulation and in sporozoites (SZ). SO7 was absent in host-derived second-stage merozoites (MZ) and was present in culture-derived first-stage MZ but at a level of only 25% of that exhibited by SZ. During invasion of Madin-Darby bovine kidney (MDBK) cells by SZ in vitro, the level of SO7 within cells, as determined by WB analysis, remained relatively constant until 48 hr of development and then decreased by about 40% at the next time point (72 hr). The SO7 secreted into the culture media during in vitro development increased to a relative maximum at 48 hr and then decreased to about 20% of maximum at 72 hr. Immunostaining with anti-rbSO7 indicates that SO7 is highly concentrated in both refractile bodies (RB) of SZ, with some limited distribution in the apical complex. Anti-rbSO7 intensively stained the intracellular parasites and the first-stage schizonts during in vitro development of E. tenella in MDBK cells. Upon release from the schizonts, the first-stage merozoites stained with 1 or 2 bright spots typically at each end. The results suggest that SO7 is closely associated with the SZ RB and is developmentally regulated but may not play a direct role in cellular invasion.

The role of rodents in the epidemiology of neosporosis was investigated by assaying brain tissue of feral mice (Mus musculus) and rats (Rattus norvegicus) for Neospora caninum. Both mouse and rat brain tissue were extracted for total DNA, and subjected to two different N. caninum-specific nested polymerase chain reaction (PCR) assays. A portion of brain tissue from the mice and rats were also assayed for N. caninum in gerbils or gamma-interferon gene knockout (KO) mice. Of the 105 feral mice tested, 10% were positive in the N. caninum-specific PCR assays. Of the 242 rats tested, 30% were positive in both assays. Although mice and rats had N. caninum by PCR testing, clinical signs of N. caninum infection were not observed nor were N. caninum parasites observed in gerbils or KO mice inoculated with the rodent brain tissue.

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.

Coccidiosis is a major intestinal disease of poultry, caused by several species of the protozoan Eimeria. The objective of this study was to examine changes in expression of digestive enzymes, nutrient transporters, and an antimicrobial peptide following an Eimeria praecox challenge of chickens at days 3 and 6 post-infection. Gene expression was determined by real-time PCR and analyzed by one-way ANOVA. In the duodenum, the primary site of E. praecox infection, a number of genes were downregulated at both d3 and d6 post-infection. These genes included liver expressed antimicrobial peptide 2 (LEAP2), the cationic (CAT1), anionic (EAAT3), and L-type (LAT1) amino acid transporters, the peptide transporter PepT1 and the zinc transporter ZnT1. Other transporters were downregulated either at d3 or d6. At both d3 and d6, there was downregulation of B(o)AT and CAT1 in the jejunum and downregulation of LEAP2 and LAT1 in the ileum. LEAP2, EAAT3, and ZnT1 have been found to be downregulated following challenge with other Eimeria species, suggesting a common cellular response to Eimeria.

SUMMARY. A method was developed to recover Eimeria spp. oocysts directly from poultry litter and determine which species of Eimeria were present using polymerase chain reaction (PCR) based on the ITS1 rDNA sequence. The species composition of Eimeria oocysts was also compared before and after propagation in susceptible chickens to determine if the relative proportion of each species changed after expansion. In samples from two broiler operations, ITS1-PCR was able to detect Eimeria spp. oocysts recovered from litter, with Eimeria acervulina, Eimeria maxima, and Eimeria praecox being the predominant species present therein. Although Eimeria tenella was found in one sample, the other species—Eimeria brunetti, Eimeria necatrix, and Eimeria mitis—were not detected. The species composition as determined by ITS1-PCR did not appear to appreciably alter after expansion in susceptible chickens. The described method represents a rapid means for determining the major Eimeria species in a poultry operation and may be helpful in choosing a particular live oocyst vaccine formulation to protect chickens against coccidiosis.

The gene for a dense granule protein (NcGRA6) of Neospora caninum was expressed in Escherichia coli as a His-tag fusion protein and purified by NiNTA affinity chromatography. In a preliminary study, high binding of antibodies from N. caninum-negative cows was observed in enzyme-linked immunosorbent assay (ELISA) using NiNTA-purified NcGRA6. Analysis of NiNTA eluates revealed a significant number of E. coli proteins that co-purified with recombinant NcGRA6. In an attempt to improve the relative sensitivity and specificity of the NcGRA6-based ELISA, the rNcGRA6 eluates were subjected to a secondary purification using reverse phase-high performance liquid chromatography (RP-HPLC). Analysis of RP-HPLC eluates by SDS-PAGE/silver staining revealed the purification of recombinant NcGRA6 from contaminating E. coli proteins. ELISAs using the RP-HPLC purified NcGRA6 (dELISA) or singly purified NcGRA6 (sELISA) for identifying seropositive and seronegative cows in a beef herd experiencing an epidemic outbreak of neosporosis were compared to standard assays based on native tachyzoite protein-immunofluorescence antibody test, immunoblot assay, and ISCOM-ELISA. The relative sensitivity, specificity, and kappa value of the NcGRA6d-ELISA were greatly improved over the NcGRA6s-ELISA when compared to the three native antigen immunoassays. These results indicate that removal of contaminating E. coli proteins improves the performance of recombinant NcGRA6 ELISA in diagnosing bovine neosporosis, and may have applicability to the use of recombinant proteins in diagnosing other infectious agents.

Gamma interferon (IFN-γ) response is essential to the development of a host protective immunity in response to infections by intracellular parasites. Neosporosis, an infection caused by the intracellular protozoan parasite Neospora caninum , is fatal when there is a complete lack of IFN-γ in the infected host. However, the mechanism by which IFN-γ is elicited by the invading parasite is unclear. This study has identified a microbial protein in the N. caninum tachyzoite N. caninum cyclophilin (NcCyP) as a major component of the parasite responsible for the induction of IFN-γ production by bovine peripheral blood mononuclear cells (PBMC) and antigen-specific CD4 + T cells. NcCyP has high sequence homology (86%) with Toxoplasma gondii 18-kDa CyP with a calculated molecular mass of 19.4 kDa. NcCyP is a secretory protein with a predicted signal peptide of 17 amino acids. Abundant NcCyP was detected in whole-cell N. caninum tachyzoite lysate antigen (NcAg) and N. caninum tachyzoite culture supernatant. In N. caninum tachyzoite culture supernatant, three NcCyP bands of 19, 22, and 24 kDa were identified. NcAg stimulated high levels of IFN-γ production by PBMC and CD4 + T cells. The IFN-γ-inducing effect of NcAg was blocked by cyclosporine, a specific ligand for CyP, in a dose-dependent manner. Furthermore, cyclosporine abolished IFN-γ production by PBMC from naïve cows as well as PBMC and CD4 + T cells from infected/immunized cows. These results indicate that the N. caninum tachyzoite naturally produces a potent IFN-γ-inducing protein, NcCyP, which may be important for parasite survival as well as host protection.

The purpose of this study was to determine whether differences in fecundity of Eimeria maxima isolates were related to their abilities to elicit cross-protective immunity. Immunizations were initiated by low-dose gavages of sporulated oocysts to day-old broiler chicks under conditions that allowed parasite recycling, and chickens were challenged with homologous and heterologous strains. Immunization efficacies were measured using a protective index calculated from weight gain, gross lesion score, plasma carotenoid, and NO 2 − + NO 3 − data. A 4×4 cross- immunization study of four E. maxima strains (designated A–D) showed that strain A, which displayed the lower fecundity, provided no cross-protection against the other three strains. Following several maintenance passages, the fecundity of strain A was increased to that of strain C, and infection with strain A oocysts was able to provide cross-immune protection against challenge with strain C. This study indicates that parasite fecundity is important in providing good immune stimulation, and should be carefully monitored when characterization of the unique immune potentials of Eimeria strains is undertaken.

Objective—To evaluate the immunologic response of a killed tachyzoite vaccine against Neospora caninum and its effectiveness in preventing fetal loss associated with experimentally induced neosporosis in sheep. Animals—30 Dorset ewes. Procedure—Ewes were randomly allocated to receive vaccination on days 1 and 60 of the study with a killed N caninum tachyzoite preparation in a commercially available adjuvant or a saline-adjuvant mixture. A ram was placed on pasture with the ewes from days 15 to 60. Blood was collected from ewes before primary and booster vaccinations and prior to experimental challenge with N caninum tachyzoite performed on day 90; sera were assessed via Neospora agglutination (NA) and immunofluorescence antibody (IFA) assays. Blood was collected from lambs before they suckled, and sera were tested for antibodies against N caninum. Results—Of the 14 vaccinated ewes that became pregnant, 12 gave birth to live-born lambs; in contrast, 5 of 11 pregnant control ewes gave birth to live-born lambs. Whereas vaccination improved fetal survival in pregnant ewes challenged with N caninum tachyzoites, it did not appear to have any appreciable effect on transmission of N caninum to offspring, as indicated by results of NA and IFA assays. Conclusions and Clinical Relevance—The N caninum tachyzoite vaccine used in this study appeared to provide protection against fetal loss associated with experimentally induced neosporosis in a high proportion of pregnant ewes. (Am J Vet Res 2004;65:1404–1408)

Isolation and biologic and molecular attributes of Neospora caninum from three littermate dogs are described. Tissue cysts were confined to the brain and striated muscles. N. caninum was isolated (isolates NC-6, NC-7, and NC-8) in rodents and cell culture that had been inoculated with brain tissue from the dogs. Schizont-like stages reactive with N. caninum antibodies were seen in cell cultures seeded with bradyzoites released from Percoll-isolated N. caninum tissue cysts from the brain of one dog. Tissue cysts were infective orally to mice and gerbils, but not to cats and dogs. The isolates were also identified as N. caninum by PCR and sequence analysis.

Objective—To evaluate the immunologic response of a killed tachyzoite vaccine against Neospora caninum and its effectiveness in preventing vertical transmission of N caninum in sheep. Animals—40 Dorset ewes seronegative for N caninum. Procedure—Group-A ewes (n = 20) were vaccinated on days 1 and 126 with a killed N caninum tachyzoite preparation in a commercially available adjuvant. Group-B ewes (n = 20) were sham vaccinated. Blood samples were collected from ewes every 2 weeks and a recombinant ELISA (rELISA) was used to determine serum antibody titers against N caninum. During pregnancy, ewes were challenged with live N caninum tachyzoites. Precolostral serum was collected from lambs and tested for antibodies against N caninum by use of an indirect fluorescence antibody test and the rELISA. Tissue specimens from stillborn lambs or lambs that died within 2 weeks of birth were collected and examined for N caninum antigen and DNA by use of immunohistochemistry and polymerase chain reaction assay, respectively. Results—Serum antibody titers against N caninum were significantly higher in group-A ewes, compared with group B ewes, following vaccination. Serum antibodies against N caninum were detected in 100% (33/33) of group-B lambs and 75% (18/24) of group-A lambs. In tissue specimens, N caninum DNA was detected in 9 of 11 group-B lambs and 0 of 10 group- A lambs. Histologically, N caninum tachyzoites were observed in 4 group-A lambs and 3 group-B lambs. Conclusions and Clinical Relevance—The killed tachyzoite vaccine against N caninum stimulated a humoral immune response in sheep and provided partial protection against vertical transmission. (Am J Vet Res 2003;64:449–452)

The purpose of this study was to characterize Eimeria maxima immune-mapped protein 1 (IMP1) that is hypothesized to play a role in eliciting protective immunity against E. maxima infection in chickens. RT-PCR analysis of RNA from unsporulated and sporulating E. maxima oocysts revealed highest transcription levels at 6-12h of sporulation with a considerable downregulation thereafter. Alignment of IMP1 coding sequence from Houghton, Weybridge, and APU-1 strains of E. maxima revealed single nucleotide polymorphisms that in some instances led to amino acid changes in the encoded protein sequence. The E. maxima (APU-1) IMP1 cDNA sequence was cloned and expressed in 2 different polyHis Escherichia coli expression vectors. Regardless of expression vector, recombinant E. maxima IMP1 (rEmaxIMP1) was fairly unstable in non-denaturing buffer, which is consistent with stability analysis of the primary amino acid sequence. Antisera specific for rEmaxIMP1 identified a single 72 kDa protein or a 61 kDa protein by non-reducing or reducing SDS-PAGE/immunoblotting. Immunofluorescence staining with anti-rEmaxIMP1, revealed intense surface staining of E. maxima sporozoites, with negligible staining of merozoite stages. Immuno-histochemical staining of E. maxima-infected chicken intestinal tissue revealed staining of E. maxima developmental stages in the lamnia propia and crypts at both 24 and 48 h post-infection, and negligible staining thereafter. The expression of IMP1 during early stages of in vivo development and its location on the sporozoite surface may explain in part the immunoprotective effect of this protein against E. maxima infection.

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.

The purpose of this study was to compare different assays for viable Cryptosporidium parvum incubated in water at a temperature commonly found in the environment. C. parvum oocysts were stored in sterile water for 9 months at 15 degrees C. A sample was removed monthly and analyzed by five different assays to determine oocyst viability. Mouse infection and cell culture showed that C. parvum oocysts remained viable and infectious when stored for 7 months at this temperature. Fluorescence in situ hybridization (FISH) using probes directed to ribosomal RNA was also applied to these oocysts. The proportion of FISH-positive oocysts was 70-80% for the first 2 months of storage, decreased and remained nearly constant at 40-50% for 3-7 months, then decreased to 20% by 8 months, and to 0% by 9 months. Amylopectin content and mRNA for amyloglucosidase (CPAG), as measured by RT-PCR, decreased much more rapidly. By 3 months and for the remainder of the incubation period, amylopectin content was 20% of the original amount present in the oocysts. The CPAG RT-PCR signal at 3 months was 50% of that observed after 1 month storage, 20% at 4 months, and was not detected thereafter. Thus, results from cell culture and mouse infection assay exhibited the best agreement, the FISH assay showed modest agreement with these assays, and CPAG RT-PCR and the amylopectin assay displayed marginal agreement with the other three assays.

Neospora caninum is a protozoan parasite of animals, which before 1984 was misidentified as Toxoplasma gondii. Infection by this parasite is a major cause of abortion in cattle and causes paralysis in dogs. Since the original description of N. caninum in 1988, considerable progress has been made in the understanding of its life cycle, biology, genetics and diagnosis. In this article, the authors redescribe the parasite, distinguish it from related coccidia, and provide accession numbers to its type specimens deposited in museums. Published by Elsevier Science Ltd. on behalf of Australian Society for Parasitology Inc.

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.

The epithelial lining of the airway tract and allergen-specific IgE are considered essential controllers of inflammatory responses to allergens. The human low affinity IgE receptor, CD23 (FcεRII), is capable of transporting IgE or IgE-allergen complexes across the polarized human airway epithelial cell (AEC) monolayer in vitro. However, it remains unknown whether the CD23-dependent IgE transfer pathway in AECs initiates and facilitates allergic inflammation in vivo, and whether inhibition of this pathway attenuates allergic inflammation. To this end, we show that in wild-type (WT) mice, epithelial CD23 transcytosed both IgE and ovalbumin (OVA)-IgE complexes across the airway epithelial barrier, while neither type of transcytosis was observed in CD23 knockout (KO) mice. In chimeric mice, OVA sensitization and aerosol challenge of WT/WT (bone-marrow transfer from the WT to WT) or CD23KO/WT (CD23KO to WT) chimeric mice, which express CD23 on radioresistant airway structural cells (mainly epithelial cells) resulted in airway eosinophilia, including collagen deposition and a significant increase in goblet cells, and increased airway hyperreactivity. In contrast, the absence of CD23 expression on airway structural or epithelial cells, but not on hematopoietic cells, in WT/CD23KO (the WT to CD23KO) chimeric mice significantly reduced OVA-driven allergic airway inflammation. In addition, inhalation of the CD23-blocking B3B4 antibody in sensitized WT mice before or during airway challenge suppressed the salient features of asthma, including bronchial hyperreactivity. Taken together, these results identify a previously unproven mechanism in which epithelial CD23 plays a central role in the development of allergic inflammation. Further, our study suggests that functional inhibition of CD23 in the airway is a potential therapeutic approach with which to inhibit the development of asthma.

Avian coccidiosis is a disease caused by intestinal protozoa in the genus Eimeria. Clinical signs of coccidiosis include intestinal lesions and reduced feed efficiency and BW gain. This growth reduction may be due to changes in expression of digestive enzymes and nutrient transporters in the intestine. The objective of this study was to examine the differential expression of digestive enzymes, transporters of amino acids, peptides, sugars, and minerals, and an antimicrobial peptide in the small intestine of Eimeria acervulina-infected broilers and layers. Uninfected broilers and layers, in general, expressed these genes at comparable levels. Some differences included 3-fold and 2-fold greater expression of the peptide transporter PepT1 and the antimicrobial peptide LEAP2 (liver expressed antimicrobial peptide 2), respectively, in the jejunum of layers compared with broilers and 17-fold greater expression of LEAP2 in the duodenum of broilers compared with layers. In the duodenum of Eimeria-infected broilers and layers, there was downregulation of aminopeptidase N; sucrase-isomaltase; the neutral, cationic, and anionic amino acid transporters b(o,+)AT/rBAT, B(o)AT, CAT2, and EAAT3; the sugar transporter GLUT2; the zinc transporter ZnT1; and LEAP2. In the jejunum of infected layers there was downregulation of many of the same genes as in the duodenum plus downregulation of PepT1, b(o,+)AT/rBAT, and the y(+) L system amino acid transporters y(+) LAT1 and y(+) LAT2. In the ileum of infected layers there was downregulation of CAT2, y(+)LAT1, the L type amino acid transporter LAT1, and the sugar transporter GLUT1, and upregulation of APN, PepT1, the sodium glucose transporter SGLT4, and LEAP2. In E. acervulina-infected broilers, there were no gene expression changes in the jejunum and ileum. These changes in intestinal digestive enzyme and nutrient transporter gene expression may result in a decrease in the efficiency of protein digestion, uptake of important amino acids and sugars, and disruption of mineral balance that may affect intestinal cell metabolism and Eimeria replication.