Katarzyna B. Miska

GENOMIC ANALYSIS OF EIMERIA SPP. POPULATIONS IN RELATION TO PERFORMANCE LEVELS OF BROILER CHICKEN FARMS IN ARKANSAS AND NORTH CAROLINA

Abstract 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.

Antibodies to the Ventral Disc Protein δ-giardin Prevent in Vitro Binding of Giardia lamblia Trophozoites

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

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.

Improved Polymerase Chain Reaction Technique for Determining the Species Composition of Eimeria in Poultry Litter

Abstract An improved polymerase chain reaction (PCR)-based method for determining the species composition of Eimeria in poultry litter was developed by incorporating species-specific internal standards in the assay. Internal standard molecules were prepared by fusing seven different Eimeria species-specific intervening transcribed sequence 1 (ITS1) rDNA primer pairs to a non-Eimeria DNA molecule and by cloning the hybrid DNA molecules into a plasmid. The internal DNA standards were then used in Eimeria-specific ITS1 PCR, and they were found to be capable of detecting E. acervulina, E. maxima, E. praecox, and E. tenella oocysts isolated directly from poultry litter.

ANALYSIS OF TRANSCRIPTS EXPRESSED BY EIMERIA TENELLA OOCYSTS USING SUBTRACTIVE HYBRIDIZATION METHODS

To characterize the genes expressed by Eimeria tenella oocysts, the sequence of 499 expressed sequence tags (ESTs) was obtained from complementary DNA (cDNAs) enriched for transcripts expressed by unsporulated or sporulated oocysts. Of these, 225 clones were isolated from cDNA of sporulated oocysts and 274 from unsporulated oocysts. A total of 163 unique sequences were found, and the majority of these (64%) represent novel genes with no significant homology to the proteins in GenBank. Approximately half of the unique transcripts generated from sporulated oocysts are also expressed by sporozoites and merozoites, whereas the expression of most (79%) of the transcripts from unsporulated oocysts has not yet been detected at other stages of development. The expression of 4 transcripts obtained from the subtracted cDNAs was confirmed by quantitative reverse transcriptase–polymerase chain reaction. The results confirmed that these transcripts are in fact differentially expressed between sporulated and unsporulated oocysts.

Molecular characterization and phylogenetic analysis of Eimeria from turkeys and gamebirds: implications for evolutionary relationships in Galliform birds.

Abstract In order to determine the evolutionary relationships among Eimeria species that parasitize birds of the Galliformes, the 18s rDNA gene and a portion of the cytochrome oxidase subunit 1 (cox-1) were amplified from Eimeria species isolated from turkeys, chukars, and pheasants. The phylogenetic analysis of these sequences suggests that species infecting chickens are polyphyletic and, therefore, do not all share a direct common ancestor. Both the 18s rDNA and the cox-1 sequences indicate that Eimeria tenella and Eimeria necatrix are more closely related to Eimeria of turkeys and pheasants than to other species that infect the chicken. It is, therefore, likely that the chicken Eimeria spp. represent 2 separate ancestral colonizations of the gut, one of which comprises E. tenella and E. necatrix that infect the ceca, while the other includes Eimeria acervulina, Eimeria brunetti, Eimeria maxima, and Eimeria mitis, which infect the upper regions of the intestine.

Application of Polymerase Chain Reaction Based on ITS1 rDNA to Speciate Eimeria

Abstract 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.

A conserved 19-KDA Eimeria tenella antigen is a profilin-like protein

A wide range of recombinant proteins from Eimeria species have been reported to offer some degree of protection against infection and disease, but the specific biological function of these proteins is largely unknown. Previous studies have demonstrated a 19-kDa protein of unknown function designated SZ-1 in sporozoites and merozoites of Eimeria acervulina that can be used to confer partial protection against coccidiosis. Reverse transcriptase–polymerase chain reaction indicated that the gene for SZ-1 is expressed by all the asexual stages of Eimeria tenella. Rabbit antisera to recombinant SZ-1 recognized an approximately 19-kDa protein from extracts of E. tenella sporozoites, merozoites, sporulated oocysts, and oocysts in various stages of sporulation. Immunofluorescence antibody staining indicated specific staining of E. tenella sporozoites and merozoites. Staining was most intense in the cytoplasm of the posterior end of the parasite. The primary amino acid sequence of the gene for E. tenella SZ-1 deduced from the E. tenella genome indicated a conserved domain for the actin-regulatory protein profilin. A conserved binding site for poly-l-proline (PLP), characteristic of profilin was also observed. SZ-1 was separated from soluble extract of E. tenella proteins by affinity chromatography using a PLP ligand, confirming the ability of SZ-1 to bind PLP. SZ-1 also partially inhibited the polymerization of actin. The current results are consistent with the classification of SZ-1 as a profilin-related protein.

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.

Cross protection studies with Eimeria maxima strains

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 NO2− + NO3− 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.