John R. Barta

The conceptual basis for a new classification of the coccidia

At the joint meeting of the 8th International Coccidiosis Conference and the Annual Scientific Meeting of the Australian Society for Parasitology in Palm Cove, Australia, in July 2001, a Controversial Roundtable was held on New classification of coccidia. The aim of this Roundtable was to stimulate and encourage discussion and debate on current classification schemes for the group of parasitic protozoa known as the eimeriid coccidia. In the past, such classifications have been based only on phenotypic characters such as morphology, ultrastructure, life cycles, and host specificity. However, over the past 10-15 years, molecular phylogenetic studies on taxa of the eimeriid coccidia have revealed that several of the families, subfamilies, and genera that have been erected based on non-molecular characters are paraphyletic. Therefore, this Roundtable was an important forum for initial discussions on how a new and more comprehensive classification of the eimeriid coccidia, which takes into consideration both phenotypic and molecular characters, can be devised. The stimulus came from invited speakers who gave introductions into selected areas of taxonomy and classification. Following these introductions, a more general discussion with the audience addressed potential steps that may be taken in future work. This review is the immediate outcome of the Roundtable. It describes advantages and disadvantages of the use of phenotypic or molecular characters as the base for taxonomic schemes for eimeriid coccidia. It gives specific examples for drawbacks of current classifications based only on phenotypic characters as well as potential pitfalls associated with the use of only molecular phylogenies. It addresses current controversies as well as rules of taxonomy and nomenclature relevant for the eimeriid coccidia. Finally, it recommends the establishment of an international group of scientists to meet on a regular basis, stimulate further discussions, and give direction on how the final goal, i.e. a proposal for a revised, and widely accepted, classification of the eimeriid coccidia, may be achieved.

Species and strain differentiation ofEimeria spp. of the domestic fowl using DNA polymorphisms amplified by arbitrary primers

Eimeria spp. from the domestic fowl were examined for genetic relatedness by the random amplified polymorphic DNA (RAPD) assay. Nine different oligonucleotide decamers with arbitrary DNA sequences were tested as primers to amplify DNA from sixEimeria species infecting chickens. Two strains each ofE. acervulina andE. tenella were used. Depending on the species/strain-primer combination, between 1 and 12 DNA segments ranging in size from 0.16 to 4.95 kb were amplified. The two strains ofE. acervulina showed minor and major differences in their amplified DNA patterns, giving a similarity coefficient of 61%. The two strains ofE. tenella seemed to be more closely related, yielding a similarity coefficient of 98%. The differences observed between species were greater than those found between strains with every primer used, indicating that the RADP assay could be a useful tool for the study of relationships among these coccidia. The results obtained in this study also indicate the presence of unique, species-specific, amplified DNA segments that could be exploited to identifyEimeria species of the chicken.

Molecular approaches for inferring evolutionary relationships among protistan parasites

Within the diverse group of parasites broadly recognized as protists, there are limited morphological characters that can be used to distinguish species and even fewer characters that can infer evolutionary relationships among species. For this reason, molecular data are commonly used to infer relationships among species and strains. These studies most commonly rely on sequences associated with the ribosomal RNA genes but increasingly other nuclear, mitochondrial or plastid genes are contributing data. Molecular systematics has been invaluable in expanding the range of characters that are available for inferring relationships among protistan taxa. As an adjunct to morphological characters, sequence data allow us to better understand the evolutionary history of this group of parasites and thereby erect a well-supported taxonomic framework that reflects these historical relationships. Importantly, the predictive nature of such a framework can aid the search for therapeutic compounds (e.g. via shared biochemical pathways) and highlight organisms that should be tested for cross-reactivity in immunological or molecular diagnostic methods (e.g. use of the closest relatives to assess test specificity). For emergent and poorly known parasites, molecular characterization and placement within the broader phylogenetic framework can help predict likely life history traits, including possible or likely definitive hosts.

Complete genomic sequence of turkey coronavirus.

n Abstractn n Turkey coronavirus (TCoV), one of the least characterized of all known coronaviruses, was isolated from an outbreak of acute enteritis in young turkeys in Ontario, Canada, and the full-length genomic sequence was determined. The full-length genome was 27,632 nucleotides plus the 3′ poly(A) tail. Two open reading frames, ORFs 1a and 1b, resided in the first two thirds of the genome, and nine additional downstream ORFs were identified. A gene for hemagglutinin-esterase was absent in TCoV. The region between the membrane (M) and nucleocapsid (N) protein genes contained three potential small ORFs: ORF-X, a previously uncharacterized ORF with an associated putative TRS within the M gene (apparently shared among all group III coronaviruses), and previously described ORFs 5a and 5b. The TCoV genome is organized as follows: 5′ UTR – replicase (ORFs 1a, 1b) – spike (S) protein – ORF3 (ORFs 3a, 3b) – small envelop (E or 3c) protein – membrane (M) protein – ORF5 (ORFs X, 5a, 5b) – nucleocapsid (N) protein −3′ UTR – poly(A). TCoV genome structure and sequence was most similar, but distinct from, avian infectious bronchitis virus (IBV). This is the first complete genome sequence for a TCoV and confirms that TCoV belongs to group III coronaviruses.n n

Immunomodulatory Effects of Feed-Borne Fusarium Mycotoxins in Chickens Infected with Coccidia:

The potential for Fusarium mycotoxins to modulate immunity was studied in chickens raised to 10 weeks of age using an enteric coccidial infection model. Experimental diets included: control, diets containing grains naturally contaminated with Fusarium mycotoxins, and diets containing contaminated grains + 0.2% polymeric glucomannan mycotoxin adsorbent (GMA). Contaminated diets contained up to 3.8 μg/g deoxynivalenol (DON), 0.3 μg/g 15-acetyl DON and 0.2 μg/g zearalenone. An optimized mixture (inducing lesions without mortality) of Eimeria acervulina, E. maxima and E. tenella was used to challenge birds at 8 weeks of age. Immune parameters were studied prior to challenge, at the end of the challenge period (7 days post-inoculation, PI), and at the end of the recovery period (14 days PI). Total serum immunoglobulin (Ig) A and IgG concentrations in challenged birds fed the contaminated diet were higher than controls at the end of the challenge period. Serum concentration of IgA, but not IgG, was significantly decreased at the end of the recovery period in birds fed the contaminated diet. The percentage of CD4+ and CD8+ cell populations in blood mononuclear cells decreased significantly at the end of the challenge period in birds fed the control or the contaminated diet compared to their percentages prior to challenge. The pre-challenge percentage of CD8+ population was restored at the end of the recovery period only in birds fed the control diet. Interferon-γ (IFN-γ) gene expression in caecal tonsils was up-regulated in challenged birds fed the contaminated diet at the end of the challenge period. No significant effect of diet was observed on oocyst counts despite the changes in the studied immune parameters. It was concluded that Fusarium mycotoxins modulate the avian immune system. This modulation involves alteration of gene expression but apparently does not enhance susceptibility or resistance to a primary coccidial challenge.

Sequencing the complete mitochondrial genome of Eimeria mitis strain USDA 50 (Apicomplexa: Eimeriidae) suggests conserved start positions for mtCOI- and mtCOIII-coding regions

Four complete mitochondrial (mt) sequences from a single-oocyst-derived line of Eimeria mitis USDA 50 were obtained (three from cloned whole-genome PCR products, one from directly sequenced whole-genome PCR product). The mt genome is 6,408xa0bp long with three genes (CytB, cytochrome c oxidase subunit I (COI) and cytochrome c oxidase subunit III (COIII)) and many rDNA fragments (large subunit rDNA 13, small subunit rDNA 10); organisation was identical to other Eimeria sp. mt genomes. Conserved start codon positions for both COI and COIII are suggested for all Eimeria mt genomes; these start codon positions exist and may also be conserved, in related apicomplexan parasites. Within the three separate cloned PCR products of near-complete mt genomes, there were 26 nucleotide differences (collectively) compared to the directly sequenced mt genome. These changes appear to be base misincorporations during PCR. Direct sequencing of long PCR amplification products may be more likely to generate accurate mt genomic sequences than cloning and subsequent sequencing.

Molecular phylogenetics of eimeriid coccidia (Eimeriidae, Eimeriorina, Apicomplexa, Alveolata): A preliminary multi-gene and multi-genome approach

Coccidia possess three distinct genomes: nuclear, mitochondrial, and plastid. Sequences from five genes located on these three genomes were used to reconstruct the phylogenetic relationships of members of the phylum Apicomplexa: 18S rDNA sequences from the nuclear (nu) genome, partial cytochrome c oxidase subunit I sequences from the mitochondrial (mt) genome, and partial 16S and 23S rDNA sequences and RNA polymerase B sequences from plastid (pl) genomes. Maximum parsimony, maximum likelihood, and Bayesian inference were used in conjunction with nuclear substitution models generated from data subsets in the analyses. Major groups within the Apicomplexa were well supported with the mitochondrial, nuclear, and a combination of mitochondrial, nuclear and concatenated plastid gene sequences. However, the genus Eimeria was paraphyletic in phylogenetic trees based on the nuclear gene. Analyses using the individual genes (18S rDNA and cytochrome c oxidase subunit I) resolved the various apicomplexan groups with high Bayesian posterior probabilities. The multi-gene, multi-genome analyses based on concatenated nu 18S rDNA, pl 16S, pl 23S, pl rPoB, pl rPoB1, and mt COI sequences appeared useful in resolving phylogenetic relationships within the phylum Apicomplexa. Genus-level relationships, or higher, appear best supported by 18S rDNA analyses, and species-level analyses are best investigated using mt COI sequences; for parasites for which both loci are available, nuclear 18S rDNA sequences combined with mitochondrial COI sequences provide a compact and informative molecular dataset for inferring the evolutionary relationships taxa in the Apicomplexa.

Seroprevalence of Turkey Coronavirus in North American Turkeys Determined by a Newly Developed Enzyme-Linked Immunosorbent Assay Based on Recombinant Antigen

ABSTRACT Turkey coronavirus (TCoV) causes diarrhea in young turkey poults, but little is known about its prevalence in the field. To address this, the complete nucleocapsid gene was cloned and expressed in Escherichia coli. Expressed nucleocapsid gene produced two distinct proteins (52 and 43 kDa); their specificity was confirmed by Western blotting using two different monoclonal antibodies. Recombinant N protein was purified and used as an antigen to develop an enzyme-linked immunosorbent assay (ELISA) for the serological detection of TCoV that was then validated using experimentally derived turkey serum. The N-based ELISA showed (97%) sensitivity and (93%) specificity for TCoV, which was significantly higher than an infectious bronchitis coronavirus-based commercial test for TCoV. To assess the utility of this recombinant ELISA, 360 serum samples from turkey farms in Ontario, Canada, and 81 serum samples from farms in Arkansas were tested for TCoV-specific antibodies. A high seroprevalence of TCoV was found in turkeys from the Ontario farms with 73.9% of breeders and 60.0% of meat turkeys testing seropositive using the N-based ELISA. Similarly, a high field prevalence was found in the turkeys from Arkansas, for which 64.2% of the serum samples tested seropositive.

Effects of feed-borne Fusarium mycotoxins and an organic mycotoxin adsorbent on immune cell dynamics in the jejunum of chickens infected with Eimeria maxima

An experiment was conducted to explore the effects of Fusarium mycotoxins, common animal feed contaminants, on intestinal immune responses to coccidia (Eimeria) in chickens. Effects of feed-borne Fusarium mycotoxins and a polymeric glucomannan mycotoxin adsorbent (GMA) on immune cell populations were studied in the jejunum of broiler breeder pullets using an Eimeria maxima infection model. Birds were fed a control diet, a diet naturally contaminated with Fusarium mycotoxins, contaminated diet plus 0.2% GMA, or control diet plus 0.2% GMA. Contaminated diets contained up to 6.5μg/g deoxynivalenol (DON), 0.47μg/g 15-acetyl-DON and 0.73μg/g zearalenone. Birds received a primary oral inoculation (1000 oocysts/bird) with E. maxima USDA strain 68 at 2 weeks of age and a secondary oral inoculation (30,000 oocysts/bird) with the same strain at 4 weeks of age. Diet-related differences in CD4(+) cell, CD8(+) cell and macrophage recruitment pattern into the jejunum were observed following both the primary and secondary infections. It was concluded that feed-borne Fusarium mycotoxins and GMA have the potential to modulate immune response to coccidial infections.

Differential display analysis of gene expression in two immunologically distinct strains of Eimeria maxima

Gene expression during sporulation and sporozoite excystation of two strains of Eimeria maxima was analyzed using the mRNA differential display technique. The two strains, the Guelph strain (GS) and a single sporocyst-derived strain (M6) from Florida, have been shown to be immunologically distinct. We isolated and cloned a 453-bp complimentary DNA (cDNA) fragment (GS-453) found only in GS sporozoites. In GS, this mRNA begins to be expressed during the earliest stages of oocyst sporulation and is continuously expressed up to and including in the excysted sporozoite. In all Northern blots, digoxigenin (DIG)-labeled GS-453 probe recognized an mRNA of approximately 1.6xa0kb from GS but not from RNA of M6. Southern blots using various endonucleases and probed with DIG-labeled GS-453 demonstrated that the genomes of both strains contained sufficiently similar sequences to permit hybridization with the probe, but the pattern of hybridization differed between the two strains. Extensive searches of the GenBank, European Molecular Biology Laboratory, and various apicomplexan expressed sequence tag databases using the DNA or inferred amino acid sequences of GS-453 cDNA clone did not identify similarity to any existing sequences.