Matthew S. Tucker

Enteric coccidian parasites harm agriculture and human health. Infectious, sporulated parasites eventually senesce. Here, we examined transcriptional changes in sporulated oocysts of Eimeria acervulina stored for 4–30 months at 4 °C. Precipitous decline in RNA abundance and transcription followed an interval of stability. Sixty constitutively expressed genes each contributed > 1000 transcripts per million (TPM) throughout, including a serine protease inhibitor, surface antigen genes, a cation-transporting ATPase, an oocyst wall protein, a zinc finger DHHC domain-containing protein, and highly expressed hypothetical proteins with no known function. Strikingly, ~82% of 6867 annotated genes underwent differential expression when comparing freshly sporulated parasites to those held for 30 months; nearly one-third of these underwent significant expression change. In freshly sporulated oocysts, 86 significantly DEGs exceeded 1000 TPM; these encoded heat shock proteins, lactate dehydrogenase, glucose-6 isomerase, and various hypothetical proteins. The oldest parasites expressed 66 DEGs, including many ribosomal subunits, a haloacid dehalogenase-like hydrolase domain-containing protein, and various hypothetical proteins. Taken together, these findings helped us to identify markers of mature parasites that remain relatively abundant in the transcript pool as oocysts age and identify other transcripts (e.g., ribosomal RNA) that increase in their relative abundance even as RNA abundance declines in senescent parasites.

Apicomplexan parasites are single-celled obligate intracellular eukaryotic organisms that cause significant animal and human disease and pose a substantial health and socioeconomic burden worldwide. Eimeria acervulina is one such parasite of chickens, representative of several Eimeria species causing coccidiosis disease. A complete assembly of the E. acervulina genome may help identify markers of drug-resistance and design recombinant vaccines. We sequenced E. acervulina APU1 strain using Oxford Nanopore Sequencing and Illumina technology in combination with a Hi-C (Omni-C) proximity linkage library and achieved a chromosomal scale assembly using the MaSuRCA assembler. The final assembly was 52 Mb. with 15 chromosomes and 99% BUSCO completeness. A total of 7,621 genes were predicted using a pipeline of BRAKER3, GeneMark-ETP and AUGUSTUS, of which 4,647 (60.97%) have a predicted Pfam function and 1,962 (25.74%) have Gene Ontology (GO) terms matching molecular, biological, and functional classes. Stage-specific transcriptome analysis revealed 9,761 transcripts. This genome assembly and transcriptome analysis provides the foundation for identifying biologically important candidates for anticoccidial drug and vaccine development.

Uso de la secuenciación profunda de amplicones basada en el gene de la citocromo oxidasa I para cuantificar los porcentajes relativos de ooquistes de Eimeria spp. en las camas avícolas. El propósito de este estudio fue evaluar un enfoque de secuenciación profunda de amplicones para estimar la abundancia relativa de diferentes ooquistes de Eimeria spp. en la cama de granjas comerciales de pollos de engorde que pueden o no estar experimentando infecciones de enteritis necrótica (EN). Se utilizaron iniciadores oligonucleotídicos dirigidos al gene mitocondrial de la citocromo oxidasa I (COI), una secuencia conservada en todas las especies de Eimeria en pollos, para amplificar mediante PCR el ADN de ooquistes de Eimeria acervulina, Eimeria maxima y Eimeria tenella. La amplificación del gene de la citocromo oxidasa I se aplicó a muestras que contenían una sola especie de Eimeria o una mezcla con partes iguales de ooquistes de E. acervulina, E. maxima y E. tenella. La secuenciación de amplicones y el mapeo de las secuencias relevantes del gene de la citocromo oxidasa I en la base de datos GenBank confirmaron el mapeo esperado de ∼100% a la Eimeria sp. apropiada y en porcentajes aproximadamente iguales (∼33%) para mezclas con números iguales de ooquistes de Eimeria spp. Este enfoque se aplicó luego al ADN derivado de ooquistes de Eimeria obtenidos a las 0, 2 y 4 semanas después de la colocación de los pollitos (crecimiento) de un total de 20 casetas individuales en seis granjas comerciales diferentes de pollos de engorde. De las siete Eimeria spp. que se sabe que infectan pollos, solo cinco se encontraron consistentemente en la cama en cada punto de tiempo de recolección: E. acervulina, E. maxima, E. mitis, Eimeria praecox y E. tenella. Los números relativos de E. maxima y de Eimeria diferentes de E. máxima (Eamipt) en todas las muestras de cama, estimados mediante secuenciación profunda de amplicones de la citocromo oxidasa I, mostraron una correlación moderada con los respectivos recuentos de ooquistes de E. maxima o Eamipt (R ∼ 0.30). Los resultados revelaron un fenómeno interesante que respalda el papel de E. mitis en la predisposición de los pollos a la enteritis necrótica. En este estudio, el porcentaje de E. mitis, estimado mediante secuenciación profunda de amplicones a las 0, 2 y 4 semanas de engorde, mostró una fuerte correlación positiva con la incidencia de enteritis necrótica (0 semanas, R = 0.57; 2 semanas, R = 0.52; 4 semanas, R = 0.61). Este estudio demuestra la utilidad de un enfoque de secuenciación profunda de amplicones para estimar la abundancia relativa de diferentes ooquistes de Eimeria que infectan pollos, ya que permite realizar las reacciones en un solo tubo, evitando así los análisis de PCR de la región espaciadora transcrita interna 1 (ITS1) específicos de cada especie, que requieren mucho tiempo y esfuerzo. Más importante aún, permite explorar las relaciones entre la incidencia de enteritis necrótica y la abundancia de especies menores de Eimeria, que se habrían pasado por alto mediante el recuento de ooquistes o la PCR de zona espaciadora transcrita interna 1, ya que la mayoría de las especies de Eimeria no son distinguibles mediante microscopía, y la PCR de la región espaciadora transcrita interna 1 no es cuantitativa.

Recurring outbreaks of cyclosporiasis linked to fresh produce demonstrate the need to develop interventions to reduce C. cayetanensis in irrigation water. C. cayetanensis is resistant to commonly used irrigation water treatments, such as chemical sanitizers, making removal of oocysts by filtration the most suitable intervention. This study evaluated the reduction of Eimeria tenella and E. acervulina, as surrogates for C. cayetanensis, in water using filters packed with sand alone or mixtures of sand and zero-valent iron (ZVI). Water inoculated with Eimeria spp. oocysts was filtered through laboratory-scale (PVC column) and field-scale (swimming pool filter) filters packed with either 100% sand or 50% ZVI/50% sand (v/v). Filtered and backflush water was examined microscopically for oocysts. Laboratory-scale filters with 50% ZVI significantly (p < 0.05) reduced 99.9% of E. tenella oocysts compared to 55.3% with filters containing 100% sand. At the field-scale level, 50% ZVI filters significantly (p < 0.05) reduced 70.5% of E. acervulina oocysts compared to 54.5% by 100% sand filters. Filters were backflushed to examine the recovery of these parasites during routine filter-media cleaning procedures. Backflush recovery of oocysts ranged from 4.42–16.7%. The addition of ZVI significantly improved the reduction of Eimeria spp. oocysts at both filter scales. and should be further investigated as a potential irrigation water intervention to reduce C. cayetanensis.

Eimeria maxima (APU1 and APU2) differ in virulence for chickens, due in part to the greater fecundity of the former. In a previous study, RNA-seq was used to identify a transcripts upregulated in E. maxima APU1 compared to E. maxima APU2. In this study, 2 of these upregulated genes (EMWEY 23530 and EMWEY 48910) were characterized by first confirming upregulation using quantitative RT-PCR. For both EMWEY 23530 and EMWEY 48910, RNA transcription was fairly consistent during sporulation. The extent of differential expression was about 2-fold log₂ higher in APU-1 compared to APU-2 (peaking at 18 h for EMWEY 23530 and 0 h for EMWEY 48910). EMWEY 23530 and EMWEY 48910 cDNA were cloned and expressed as polyHis-fusion proteins in Escherichia coli. The observed size of recombinant EMWEY 23530 was 24 kDa; the observed size of recombinant EMWEY 48910 was 35 kDa, which are consistent with the predicted size based on the coding sequences. Immunostaining 2D gel blots of E. maxima APU1 and APU2 oocyst/sporocyst protein with antisera specific for EMWEY 23530 identified a 33.5 kDa protein with a pH 7.4 isoelectric point (Emax p33.5). Similar 2D gel blot analysis with EMWEY 48910 identified a 41 kDa protein with a pH 7.2 isoelectric point (Emax p41). The intensity of Emax p33.5 and Emax p41 was noticeably greater in oocyst/sporocyst proteins from E. maxima APU1 compared to E. maxima APU2. This was corroborated by ELISA wherein equal amounts of total E. maxima APU1 and APU2 protein were probed with serial dilutions of anti-rEmax p33.5 or anti-rEmax p41. Immunofluorescence (IFA) staining of permeabilized unsporulated E. maxima APU1 and APU2 oocysts revealed Emax p33.5 to be localized in one end of oocysts, while Emax p41 appeared on the surface of oocysts. After sporulation, the p33.5 and p41 antigens appeared loosely associated with sporocysts. Taken together, these data confirm excess expression of two proteins in the E. maxima strain characterized by greater fecundity and virulence, and may provide insight into basis for phenotypic differences among different E. maxima.

Giardia duodenalis, a major cause of waterborne infection, infects a wide range of mammalian hosts and is subdivided into eight genetically well-defined assemblages named A through H. However, fragmented genomes and a lack of comparative analysis within and between the assemblages render unclear the molecular mechanisms controlling host specificity and differential disease outcomes. To address this, we generated a near-complete de novo genome of AI assemblage using the Oxford Nanopore platform by sequencing the Be-2 genome. We generated 148,144 long-reads with quality scores of > 7. The final genome assembly consists of only nine contigs with an N50 of 3,045,186 bp. This assembly agrees closely with the assembly of another strain in the AI assemblage (WB-C6). However, a critical difference is that a region previously placed in the five-prime region of Chr5 belongs to Chr4 of Be-2. We find a high degree of conservation in the ploidy, homozygosity, and the presence of cysteine-rich variant-specific surface proteins (VSPs) within the AI assemblage. Our assembly provides a nearly complete genome of a member of the AI assemblage of G. duodenalis, aiding population genomic studies capable of elucidating Giardia transmission, host range, and pathogenicity.

For reasons unknown, Eimeria maxima is unique among Eimeria species infecting chickens in the immunovariability it displays among isolates from different geographical areas. Eimeria maxima oocysts (named EmaxAPU3) were isolated late in grow-out (6 weeks) from litter in a commercial broiler operation that was using Eimeria vaccination as the coccidiosis control program. Cross-protection studies (n = 4) were conducted in immunologically naïve chickens between EmaxAPU3 and two E. maxima lab strains (EmaxAPU1, EmaxAPU2) by immunizing with one E. maxima strain and challenging with either the homologous or heterologous E. maxima. As measured by oocyst output, immunization with EmaxAPU1 protected against homologous challenge (EmaxAPU1) and against heterologous challenge with EmaxAPU3, but not against EmaxAPU2. Similarly, immunization with EmaxAPU3 protected against homologous challenge (EmaxAPU3) and against heterologous challenge with EmaxAPU1, but not against EmaxAPU2. Immunization of chickens with EmaxAPU2 elicited a protective response against homologous challenge (EmaxAPU2), but not against EmaxAPU1 nor EmaxAPU3. The most plausible explanation for the appearance of this immunovariant late in grow-out is that E. maxima APU3 escaped immunity directed to E. maxima antigenic types in the commercial vaccine.

Eimeria tenella is a major cause of caecal coccidiosis in commercial poultry chickens worldwide. Here, we report chromosomal scale assembly of Eimeria tenella strain APU2, a strain isolated from commercial broiler chickens in the U.S. We obtained 100× sequencing Oxford Nanopore Technology (ONT) and more than 800× Coverage of Illumina Next-Seq. We created the assembly using the hybrid approach implemented in MaSuRCA, achieving a contiguous 51.34 Mb chromosomal-scale scaffolding enabling identification of structural variations. The AUGUSTUS pipeline predicted 8060 genes, and BUSCO deemed the genomes 99% complete; 6278 (78%) genes were annotated with Pfam domains, and 1395 genes were assigned GO-terms. Comparing E. tenella strains (APU2, US isolate and Houghton, UK isolate) derived Houghton strain of E. tenella revealed 62,905 high stringency differences, of which 45,322 are single nucleotide polymorphisms (SNPs) (0.088%). The rate of transitions/transversions among the SNPs are 1.63 ts/tv. The strains possess conserved gene order but have profound sequence heterogeneity in a several chromosomal segments (chr 2, 11 and 15). Genic and intergenic variation in defined gene families was evaluated between the two strains to possibly identify sequences under selection. The average genic nucleotide diversity of 2.8 with average 2 kb gene length (0.145%) at genic level. We examined population structure using available E. tenella sequences in NCBI, revealing that the two E. tenella isolates from the U.S. (E. tenella APU2 and Wisconsin, “ERR296879”) share a common maternal inheritance with the E. tenella Houghton. Our chromosomal level assembly promotes insight into Eimeria biology and evolution, hastening drug discovery and vaccine development.

Strains of Eimeria maxima, an enteric parasite of poultry, vary in virulence. Here, we performed microscopy and RNA sequencing on oocysts of strains APU-1 (which exhibits more virulence) and APU-2. Although each underwent parallel development, APU-1 initially approached maturation more slowly. Each strain sporulated by hour 36; their gene expression diverged somewhat thereafter. Candidate biomarkers of viability included 58 genes contributing at least 1000 Transcripts Per Million throughout sporulation, such as cation-transporting ATPases and zinc finger domain-containing proteins. Many genes resemble constitutively expressed genes also important to Eimeria acervulina. Throughout sporulation, the expression of only a few genes differed between strains; these included cyclophilin A, EF-1α, and surface antigens (SAGs). Mature and immature oocysts uniquely differentially express certain genes, such as an X-Pro dipeptidyl-peptidase domain-containing protein in immature oocysts and a profilin in mature oocysts. The immature oocysts of each strain expressed more phosphoserine aminotransferase and the mature oocysts expressed more SAGs and microneme proteins. These data illuminate processes influencing sporulation in Eimeria and related genera, such as Cyclospora, and identify biological processes which may differentiate them. Drivers of development and senescence may provide tools to assess the viability of oocysts, which would greatly benefit the poultry industry and food safety applications.