Raymond H. Fetterer

Identification and Characterization of Neospora caninum Cyclophilin That Elicits Gamma Interferon Production

ABSTRACT 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.

Eimeria acervulina: evaluation of the cellular and antibody responses to the recombinant coccidial antigens in B-congenic chickens

The roles of major histocompatibility complex (MHC) and non-MHC-linked genes in the genetic control of disease susceptibility and the development of protective immunity to Eimeria acervulina infection were investigated in six 15I5-B congenic and four different strains of chickens characterized for the MHC. When oocyst production was assessed, wide variations were noted following initial and challenge infections among the strains of chickens tested. In general, 15.N-21, 15.P-13, B21, B19, SC, and FP chickens were protected following challenge infection whereas 15I5, 15.P-19, 15.7-2, and 15.6-2 chickens were not. Strains of chickens sharing a same B haplotype on different genetic backgrounds did not show comparable levels of protection. These results lead to the view that non-MHC-linked genes have a profound influence on the outcome of the host response to E. acervulina infection. Chickens infected twice at 1-month intervals by an oral inoculation with E. acervulina developed both coccidial-specific antibody and T-cell responses. E. acervulina infected chickens showed T-cell-mediated immune responses to the intact sporozoites as well as to recombinant proteins, p130 of sporozoites and p150 of merozoites. Both p130 and p150 antigens have been identified and characterized previously. Sera obtained from all infected chickens recognized the p150 merozoite protein, but not the p130 sporozoite protein in immunoblots. In general, the cellular response, but not the antibody response to the p150 recombinant surface merozoite antigen correlated with the degree of protection following the challenge infection. These results suggest that the strains of chickens having improved protection against challenge infection demonstrate higher T-cell responses to the recombinant surface merozoite protein, p150.

Comparison of Eimeria Species Distribution and Salinomycin Resistance in Commercial Broiler Operations Utilizing Different Coccidiosis Control Strategies

Abstract The purpose of the present study was to evaluate the species composition and salinomycin sensitivity of Eimeria oocysts isolated from commercial broiler farms that differed by means of coccidiosis control (anticoccidial drugs [ACD] vs. live oocyst vaccines [VAC]). A comparison of Eimeria species composition and salinomycin sensitivity was also made before and after a producer switched from salinomycin to live oocyst vaccines. In general, no significant difference was observed in the concentration of Eimeria spp. oocysts in litter from VAC-utilizing farms compared to litter from ACD-utilizing farms. Application of PCR-based methods to detect coccidia found that Eimeria species distribution in litter from VAC operations more closely resembled the species composition in the live oocyst vaccines. Drug sensitivity testing found that Eimeria oocysts from VAC operations displayed greater salinomycin sensitivity as measured by weight gain and feed conversion efficiency compared to oocysts from ACD farms. These findings provide additional evidence for the usefulness of live oocyst vaccines to restore ionophore sensitivity in poultry operations that contain an ionophore-resistant population of Eimeria spp. oocysts.

Activity of Benzimidazoles against Cryptosporidiosis in Neonatal BALB/c Mice

The need for an effective compound for the prevention and treatment of cryptosporidiosis in humans and animals has led to the testing of benzimidazoles based on reports that albendazole was clinically effective against related protozoan parasites causing microsporidiosis in humans. Albendazole and other benzimidazole derivatives were tested for prophylactic efficacy against cryptosporidiosis at dosage levels 1-3x the levels found effective for treatment of cattle or sheep for helminth infections. Daily dosage levels of thiabendazole, parbendazole, oxibendazole, mebendazole, and albendazole, as high as 200, 30, 10, 15, and 15 mg/kg of body weight, respectively, were not efficacious in neonatal mice. Although the number of parasites in histologic sections of intestine from mice mediated with 15 mg albendazole/kg of body weight was significantly lower than in unmedicated control mice, suggesting activity against the parasite, a high percentage of epithelial cells in the medicated mice were infected.

Mode of Action of Antinematodal Drugs

The purpose of this chapter is to describe biochemical or physiological events observed in nematodes exposed to an anthelmintic. Whether these pharmacologically mediated events truly represent a mode of action is difficult to prove, and the action described may represent a secondary event unrelated to the mode of action or it may be part of a series of events involved in the killing process.

Expression of digestive enzymes and nutrient transporters in Eimeria acervulina-challenged layers and broilers

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.

Synthesis of tyrosine-derived cross-links in Ascaris suum cuticular proteins.

Tritiated dityrosine and isotrityrosine were detected by high performance liquid chromatography (HPLC) of acid hydrolysates of cuticular proteins from larval Ascaris suum following their 96-hr in vitro incubation in [3H]tyrosine. Sixty percent of the HPLC-recovered radiolabel was present as tyrosine, 20% as dityrosine, and 6% as isotrityrosine. Approximately 13% of radioactivity was associated with several unidentified peaks. A similar distribution of radioactivity was observed in acid hydrolysates of cuticular proteins from young adults of A. suum following 48 hr in vitro incubation with [3H]tyrosine. The 2-mercaptoethanol (2ME)-insoluble cuticular protein from the larval stages had a higher rate of synthesis of [3H]dityrosine than did the 2ME-soluble cuticular proteins, whereas the 2ME-soluble cuticular proteins had higher rates of synthesis of [3H]isotrityrosine. Pulse-chase studies of A. suum larvae demonstrated a relatively low rate of synthesis of both dityrosine and isotrityrosine. The addition to the culture media of the peroxidase inhibitors, phenylhydrazine (PHEN), 3-amino-1,2,4-triazole (AT), and N-acetyltyrosine (NAT) reduced the amount of [3H]tyrosine synthesized into both dityrosine and isotrityrosine. In a cell-free system, soluble extracts of A. suum larvae also converted radiolabeled tyrosine to dityrosine; isotrityrosine was produced by some extracts. The rate of conversion correlated with time of incubation and the volume of added extract and was inhibited by AT, NAT, and PHEN, with PHEN being the most potent inhibitor. The results of the present study suggest that the tyrosine residues of the cuticular proteins are posttranslationally modified by the formation of dityrosine and isotrityrosine cross-links. This modification is most likely mediated by a peroxidase.

Expression of digestive enzymes and nutrient transporters in Eimeria-challenged broilers.

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 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.

Neospora caninum: cloning and expression of a gene coding for cytokine-inducing profilin.

Profilins are actin-binding proteins that in Toxoplasma gondii stimulate innate immunity in mice by binding Toll-like receptors (TLR) on dendritic cells (DC) leading to release of inflammatory cytokines, primarily IL-12 and IFN-gamma. The purpose of the present study was to characterize Neospora caninum profilin, termed NcProfilin. Recombinant NcProfilin was purified by affinity chromatography, and used to prepare specific antisera to allow characterization of native NcProfilin antigen in N. caninum tachyzoites. By immunoblotting, recombinant NcProfilin is 22kDa, and is similar in size to the respective 22kDa native protein. Immunofluorescence and immunoelectron microscopy localized native NcProfilin to the apical end of N. caninum tachyzoites. Incubation of recombinant NcProfilin with spleen cells from BALB/c mice induced release of IFN-gamma. Also, injection of BALB/c mice with purified rNcProfilin elicited a strong IFN-gamma and IL-12 responses at 6 and 24h after injection indicating that NcProfilin may be an important protein in regulation of cytokine responses to N. caninum.