A Qualitative Analysis of Narrative Preclerkship Assessment Data to Evaluate Teamwork Skills.

Construct: Students entering the health professions require competency in teamwork. Background: Although many teamwork curricula and assessments exist, studies have not demonstrated robust longitudinal assessment of preclerkship students' teamwork skills and attitudes. Assessment portfolios may serve to fill this gap, but it is unknown how narrative comments within portfolios describe student teamwork behaviors. Approach: We performed a qualitative analysis of narrative data in 15 assessment portfolios. Student portfolios were randomly selected from 3 groups stratified by quantitative ratings of teamwork performance gathered from small-group and clinical preceptor assessment forms. Narrative data included peer and faculty feedback from these same forms. Data were coded for teamwork-related behaviors using a constant comparative approach combined with an identification of the valence of the coded statements as either "positive observation" or "suggestion for improvement." Results: Eight codes related to teamwork emerged: attitude and demeanor, information facilitation, leadership, preparation and dependability, professionalism, team orientation, values team member contributions, and nonspecific teamwork comments. The frequency of codes and valence varied across the 3 performance groups, with students in the low-performing group receiving more suggestions for improvement across all teamwork codes. Conclusions: Narrative data from assessment portfolios included specific descriptions of teamwork behavior, with important contributions provided by both faculty and peers. A variety of teamwork domains were represented. Such feedback as collected in an assessment portfolio can be used for longitudinal assessment of preclerkship student teamwork skills and attitudes.

Development of a metacognitive effort construct of empathy during clinical training: a longitudinal study of the factor structure of the Jefferson Scale of Empathy.

Empathy is crucial for effective clinical care but appears to decline during undergraduate medical training. Understanding the nature of this decline is necessary for addressing it. The Jefferson Scale of Empathy (JSE) is used to measure medical students' clinical empathy attitudes. One recent study described a 3-factor model of the JSE. This model was found in responses from matriculating medical students, but little is known about how the factor structure of the scale changes during clinical training. The Learning Environment Study is a longitudinal prospective study of two cohorts from 28 medical schools. At matriculation and at the end of each subsequent year, students self-reported clinical empathy attitudes using the JSE. Data from 4,797 students were randomly partitioned for exploratory (EFA) and confirmatory factor analyses using responses from preclinical and clinical years of medical school. Five models were compared for confirmatory factor analysis: two null models for control, the recent 3-factor model, and the two models resulting from the EFAs of preclinical and clinical year responses. Preclinical year responses yielded a 3-factor model similar to the recent 3-factor model. Clinical year responses yielded a 4-factor model ("feelings," "importance," "ease," and "metacognitive effort") suggesting changes in the structure of clinical empathy attitudes over time. Metacognitive effort showed the largest decline over time. The model is a better fit for both preclinical and clinical responses and may provide more insight into medical students' clinical empathy attitudes than other models. The emergence of metacognitive effort in the clinical years suggests empathy may become more nuanced for students after clinical exposure and may account for much of the observed decline in clinical empathy attitudes.

Students as Teachers: Development and Implementation of a Workplace-Based Assessment.

Students as Teachers programs are prevalent, though assessments within these programs are lacking. A workplace-based assessment for clinical teaching was developed to foster formative feedback and support learner growth. Feedback narratives were analyzed to identify student teaching behaviors and demonstrated the themes medical knowledge, professionalism, communication, and teaching skills, which were subcategorized as clinical relevance, learner stage appropriateness, use of evidence-based teaching strategies, learning environment, feedback-related, and time-appropriate. This analysis supports the use of the assessment form for student teachers in the clinical environment as students received construct-relevant feedback from various raters while teaching in multiple settings.

A Study of Cross-Protection between Eimeria maxima Immunovariants.

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.

Using Natural Language Processing to Visualize Narrative Feedback in a Medical Student Performance Dashboard.

PROBLEM: Clinical competency committees rely on narrative feedback for important insight into learner performance, but reviewing comments can be time-consuming. Techniques such as natural language processing (NLP) could create efficiencies in narrative feedback review. In this study, the authors explored whether using NLP to create a visual dashboard of narrative feedback to preclerkship medical students would improve the competency review efficiency. APPROACH: Preclerkship competency review data collected at the Northwestern University Feinberg School of Medicine from 2014 to 2021 were used to identify relevant features of narrative data associated with review outcome (ready or not ready) and draft visual summary reports of the findings. A user needs analysis was held with experienced reviewers to better understand work processes in December 2019. Dashboards were designed based on this input to help reviewers efficiently navigate large amounts of narrative data. The dashboards displayed the model's prediction of the review outcome along with visualizations of how narratives in a student's portfolio compared with previous students' narratives. Excerpts of the most relevant comments were also provided. Six faculty reviewers who comprised the competency committee in spring 2023 were surveyed on the dashboard's utility. OUTCOMES: Reviewers found the predictive component of the dashboard most useful. Only 1 of 6 reviewers (17%) agreed that the dashboard improved process efficiency. However, 3 (50%) thought the visuals made them more confident in decisions about competence, and 3 (50%) thought they would use the visual summaries for future reviews. The outcomes highlight limitations of visualizing and summarizing narrative feedback in a comprehensive assessment system. NEXT STEPS: Future work will explore how to optimize the dashboards to meet reviewer needs. Ongoing advancements in large language models may facilitate these efforts. Opportunities to collaborate with other institutions to apply the model to an external context will also be sought.

Recommendations to address and research systemic bias in assessment: perspectives from directors of research in medical education.

INTRODUCTION: Addressing systemic bias in medical school assessment is an urgent task for medical education. This paper outlines recommendations on topic areas for further research on systemic bias, developed from a workshop discussion at the 2023 annual meeting of the Society of Directors of Research in Medical Education. MATERIALS AND METHODS: During the workshop, directors engaged in small-group discussions on guidelines to address bias in assessment practices following a proposed categorization of 'Do's,' 'Don'ts,' and 'Don't knows' and listed their insights using anonymous sticky notes, which were shared and discussed with the larger group of participants. The authors performed a content analysis of the notes through deductive and inductive coding. We reviewed and discussed our analysis to reach consensus. RESULTS: The workshop included 31 participants from 28 institutions across the US and Canada, generating 51 unique notes. Participants identified 23 research areas in need of further study. The inductive analysis of proposed research areas revealed four main topics: 1) The role of interventions, including pre-medical academic interventions, medical-education interventions, assessment approaches, and wellness interventions; 2) Professional development, including the definition and assessment of professionalism and professional identity formation; 3) Context, including patient care and systemic influences; and 4) Research approaches. DISCUSSION: While limited to data from a single workshop, the results offered perspectives about areas for further research shared by a group of directors of medical education research units from diverse backgrounds. The workshop produced valuable insights into the need for more evidence-based interventions that promote more equitable assessment practices grounded in real-world situations and that attenuate the effects of bias.

Aqueous Ozone Exposure Inhibits Sporulation in the Cyclospora cayetanensis Surrogate Eimeria acervulina.

Ozone is a potent disinfecting agent used to treat potable water and wastewater, effectively clearing protozoa such as Giardia and Cryptosporidium spp. It is unclear whether ozone treatment of water or fresh produce can reduce the spread of the emerging parasite Cyclospora cayetanensis, which causes cyclosporiasis in humans. Obtaining viable C. cayetanensis oocysts to evaluate inactivation methods is challenging because we lack the means to propagate them in vitro, because of delays in case reporting, and because health departments typically add inactivating fixatives to clinical specimens. Research in various surrogate organisms has sought to bolster understanding of the biology of C. cayetanensis. Among these surrogates is the poultry parasite Eimeria acervulina, a closely related and easily cultured parasite of economic significance. We used this surrogate to evaluate the consequences of ozone treatment, using the sporulation state as an indicator of infectious potential. Treating with ozonated water acidified with citric acid reduced sporulation ability in a dose-dependent manner; treatment with up to 4.93 mg/L initial concentration of ozone resulted in a 93% inactivation of sporulation by 7 days posttreatment. This developmental arrest was accompanied by transcriptional changes in genes involved in regulating the response to reactive oxygen species (ROS) in a time course that is consistent with the production of oxygen free radicals. This study shows that ozone is highly effective in preventing sporulation of E. acervulina, a model coccidian used as a surrogate for Cyclospora. Furthermore, ozone exposure induced molecular responses to general oxidative stress, documented with several well-characterized antioxidant enzymes.

Precision Education: The Future of Lifelong Learning in Medicine.

ABSTRACT: The goal of medical education is to produce a physician workforce capable of delivering high-quality equitable care to diverse patient populations and communities. To achieve this aim amidst explosive growth in medical knowledge and increasingly complex medical care, a system of personalized and continuous learning, assessment, and feedback for trainees and practicing physicians is urgently needed. In this perspective, the authors build on prior work to advance a conceptual framework for such a system: precision education (PE).PE is a system that uses data and technology to transform lifelong learning by improving personalization, efficiency, and agency at the individual, program, and organization levels. PE "cycles" start with data inputs proactively gathered from new and existing sources, including assessments, educational activities, electronic medical records, patient care outcomes, and clinical practice patterns. Through technology-enabled analytics , insights are generated to drive precision interventions . At the individual level, such interventions include personalized just-in-time educational programming. Coaching is essential to provide feedback and increase learner participation and personalization. Outcomes are measured using assessment and evaluation of interventions at the individual, program, and organizational levels, with ongoing adjustment for repeated cycles of improvement. PE is rooted in patient, health system, and population data; promotes value-based care and health equity; and generates an adaptive learning culture.The authors suggest fundamental principles for PE, including promoting equity in structures and processes, learner agency, and integration with workflow (harmonization). Finally, the authors explore the immediate need to develop consensus-driven standards: rules of engagement between people, products, and entities that interact in these systems to ensure interoperability, data sharing, replicability, and scale of PE innovations.

Protecting chickens against coccidiosis in floor pens by administering Eimeria oocysts using gel beads or spray vaccination.

Control of avian coccidiosis is increasingly being achieved by the administration of low doses of Eimeria oocysts to newly hatched chicks. The purpose of this study was to test the efficacy of gel beads containing a mixture of Eimeria acervulina, Eimeria maxima, and Eimeria tenella oocysts as a vaccine to protect broilers raised in contact with litter. Newly hatched chicks were either sprayed with an aqueous suspension of Eimeria oocysts or were allowed to ingest feed containing Eimeria oocysts-incorporated gel beads. Control, 1-day-old chicks were given an equivalent number of Eimeria oocysts (10(3) total) by oral gavage or received no vaccine (nonimmunized controls). All chicks were raised in floor-pen cages in direct contact with litter. At 4 wk of age, all chickens and a control nonimmunized group received a high-dose E. acervulina, E. maxima, and E. tenella challenge infection. Chickens immunized with Eimeria oocysts in gel beads or by spray vaccination displayed significantly (P < 0.05) greater weight gain (WG) compared to nonimmunized controls. Feed conversion ratio (FCR) also showed a significant (P < 0.05) improvement in both groups relative to nonimmunized controls. Moreover, WG and FCR in both groups was not significantly different (P > 0.05) from chickens immunized by oral gavage or from nonimmunized, noninfected controls. Oocyst excretion after Eimeria challenge by all immunized groups was about 10-fold less than in nonimmunized controls. These findings indicate that immunization efficacy of gel beads and spray vaccination is improved by raising immunized chicks in contact with litter.

Relationship between Eimeria oocyst infectivity for chickens and in vitro excystation of E. acervulina, E. maxima, and E. tenella oocyst during long-term storage.

Vaccination of chickens against avian coccidiosis in chickens often involves storing Eimeria oocysts for months after oocyst propagation and sporulation. The purpose of this study was to determine how long E. acervulina, E. maxima, and E. tenella oocysts remained viable when stored at refrigeration (4°C) or egg room (20°C) temperatures. Separate tubes containing E. acervulina, E. maxima, or E. tenella oocysts were stored at these temperatures and a sample removed every 3 mo for inoculating chickens for evidence of a patent infection. Also, an aliquot of each Eimeria species at each time-temperature combination was subjected to in vitro excystation to quantify the relative number of released sporozoites to intact (nonexcysted) sporocysts. Eimeria tenella appeared to be most susceptible to storage in that no oocyst production was observed at 9 mo at either temperature. Although E. maxima oocysts were viable at 9 mo, no oocyst production was observed at 12 mo storage at these 2 temperatures. Quite unexpected was that E. acervulina was much more stable than E. tenella and E. maxima remaining viable up to and including 27 mo at 4°C and up to and including 12 mo at 20°C. No consistent correlation was observed between in vivo oocyst production and in vitro excystation arising from these 2 respective temperatures (E. acervulina r = 0.58, r = 0.54; E. maxima r = 0.90, r = 0.54; E. tenella r = 0.38, r = 0.90). These data indicate that attention must be paid to time and temperature of Eimeria oocyst storage, and that sporozoite excystation may not be a good indicator of oocyst viability, particularly at later timepoints in incubation.

Effects of codon optimization on expression in Escherichia coli of protein-coding DNA sequences from the protozoan Eimeria.

The objective of this study was to compare the levels of recombinant protein from three Eimeria genes before and after optimization of codons for expression in Escherichia coli. Protein coding sequences from Eimeria maxima (EmaxSO7, EmaxIMP1) and Eimeria acervulina (EAH00033530) were cloned with or without prior codon optimization and expressed as polyHis fusion proteins. All three outcomes: higher, lower, or no change in the yield of amount of recombinant protein were observed suggesting that codon optimization alone for expression in E. coli does not inevitably lead to higher expression levels. Analysis of codon usage for each gene sequence revealed that, similar to other organisms, Eimeria intersperses rare and frequently used codons in protein-coding sequences. However, no relationship was observed between the predicted protein structure and the location of major and minor codons, suggesting that codon selection in this apicomplexan parasite is influenced by factors other than regional secondary protein structure.

Utilizing Natural Language Processing of Narrative Feedback to Develop a Predictive Model of Pre-Clerkship Performance: Lessons Learned.

Background: Natural language processing is a promising technique that can be used to create efficiencies in the review of narrative feedback to learners. The Feinberg School of Medicine has implemented formal review of pre-clerkship narrative feedback since 2014 through its portfolio assessment system but this process requires considerable time and effort. This article describes how natural language processing was used to build a predictive model of pre-clerkship student performance that can be utilized to assist competency committee reviews. Approach: The authors took an iterative and inductive approach to the analysis, which allowed them to identify characteristics of narrative feedback that are both predictive of performance and useful to faculty reviewers. Words and phrases were manually grouped into topics that represented concepts illustrating student performance. Topics were reviewed by experienced reviewers, tested for consistency across time, and checked to ensure they did not demonstrate bias. Outcomes: Sixteen topic groups of words and phrases were found to be predictive of performance. The best-fitting model used a combination of topic groups, word counts, and categorical ratings. The model had an AUC value of 0.92 on the training data and 0.88 on the test data. Reflection: A thoughtful, careful approach to using natural language processing was essential. Given the idiosyncrasies of narrative feedback in medical education, standard natural language processing packages were not adequate for predicting student outcomes. Rather, employing qualitative techniques including repeated member checking and iterative revision resulted in a useful and salient predictive model.

RNA-Seq of Phenotypically Distinct Eimeria maxima Strains Reveals Coordinated and Contrasting Maturation and Shared Sporogonic Biomarkers with Eimeria acervulina.

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.

Chromosomal scale assembly reveals localized structural variants in avian caecal coccidian parasite Eimeria tenella.

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.

Gel-Bead Delivery of Eimeria oocysts protects chickens against coccidiosis.

Vaccines composed of either virulent or attenuated Eimeria spp. oocysts have been developed as an alternative to medication of feed with ionophore drugs or synthetic chemicals. The purpose of this study was to evaluate the use of gel-beads containing a mixture of Eimeria acervulina, Eimeria maxima, and Eimeria tenella oocysts as a vaccine against coccidiosis. Newly hatched chicks (Gallus gallus domesticus) were either sprayed with an aqueous suspension of Eimeria oocysts or were allowed to ingest feed containing Eimeria oocysts-incorporated gel-beads. Control day-old chicks were given an equivalent number of Eimeria oocysts (10(4) total) by oral gavage. After 3 days, chicks were randomly assigned to individual cages, and feces were collected between days 5 and 8 postinfection. All samples were processed for total Eimeria oocysts. At 4 wk of age, all chickens and a control nonimmunized group received a high-dose E acervulina, E maxima, and E. tenella challenge infection. Oocyst excretion by chicks fed gel-beads or inoculated by oral gavage was 10- to 100-fold greater than that of chicks spray-vaccinated with the Eimeria oocysts mixture (log 6.3-6.6 vs. log 4.8). Subsequent protection against challenge as measured by weight gain and feed conversion efficiency was significantly greater (P < 0.05) in gel-bead and oral gavage groups compared with spray-vaccinated or nonimmunized groups. Also, gel-bead and oral gavage groups showed no significant difference (P > 0.05) in weight gain and feed conversion efficiency compared with nonchallenged controls. These findings indicate that incorporation of Eimeria spp. oocysts in gel-beads may represent an effective way to deliver live oocyst vaccines to day-old chicks for preventing subsequent outbreaks of coccidiosis in the field.

Immunolocalization of ß- and δ-giardin within the ventral disk in trophozoites of Giardia duodenalis using multiplex laser scanning confocal microscopy.

Immunolocalization of ß- and δ-giardin in Giardia duodenalis trophozoites revealed that both giardins are strictly associated with the ventral disk (VD). Optical sectioning of the immunolabeled VD, together with quantitative colocalization of δ- and ß-giardin immunoreactivity, demonstrated that δ-giardin is primarily localized to the ventral side, and ß-giardin is localized to the dorsal side of the VD.

Polymerase Chain Reaction Directed to Eimeria ITS1 rDNA or a Single-Copy Orthologue Corroborates Standard Micro-oocyst Analysis of Intestinal Tissue from Chickens Infected with E. acervulina, E. maxima, or E. tenella.

The purpose of this study was to compare micro-oocyst counts of Eimeria to PCR analysis of intestinal DNA from smears of duodenum, jejunum/ileum, and cecum of chickens infected with Eimeria acervulina, Eimeria maxima, or Eimeria tenella oocysts. Broiler chicks were infected in triplicate with various doses of E. acervulina, E. maxima, or E. tenella oocysts and were necropsied 5-6 days later to recover duodenal, jejunal, or cecal tissue for micro-oocyst count and for DNA recovery. Micro-oocyst counts were done independently by three individuals. Micro-oocyst counts and PCR directed to ITS1 rDNA or to a single-copy orthologue (SCO 5995) displayed a linear relationship with oocyst dose for each Eimeria species. A strong correlation was found between mean micro-oocyst counts and both PCR assays for E. acervulina (r = 0.78-0.94), E. maxima (r = 0.79-0.91), and E. tenella (r = 0.85-0.96). There was good agreement between ITS1 and SCO 5995 PCR assays: E. acervulina (r = 0.92), E. maxima (r = 0.79), and E. tenella (r = 0.93). However, only ITS1 PCR analysis corroborated micro-oocyst counts of Eimeria oocyst DNA recovered from Eimeria-infected broiler chickens submitted to a poultry diagnostic laboratory. These findings suggest that ITS1 PCR or SCO PCR can validate traditional micro-oocyst counts used in quantifying Eimeria infection in chickens. Additional studies may provide a method for estimating the relative abundance of each Eimeria species in a natural infection.

Reacción en cadena de la polimerasa dirigida al gene ITS1 rDNA de Eimeria o a un ortólogo de copia única corrobora el análisis estándar de microquistes del tejido intestinal de pollos infectados con E. acervulina, E. maxima o E. tenella. El propósito de este estudio fue comparar los recuentos de micro-ooquistes de Eimeria con el análisis PCR del ADN intestinal de frotis de duodeno, yeyuno/íleon y ciego de pollos infectados con ooquistes de Eimeria acervulina, Eimeria maxima o Eimeria tenella. Los pollos de engorde se infectaron por triplicado con varias dosis de ooquistes de E. acervulina, E. maxima o E. tenella y se les realizó la necropsia entre los cinco y seis días después para recuperar tejido duodenal, yeyunal o cecal para el recuento de micro-ooquistes y para la recuperación de ADN. Los recuentos de micro-ooquistes se realizaron de forma independiente por tres personas. Los recuentos de micro-ooquistes y la PCR dirigida al gene ITS1-rDNA o a un ortólogo de copia única (SCO 5995) mostraron una relación lineal con la dosis de ooquistes para cada especie de Eimeria. Se encontró una fuerte correlación entre el recuento medio de micro-ooquistes y ambos ensayos de PCR para E. acervulina (r = 0.78­0.94), E. maxima (r = 0.79­0.91) y E. tenella (r = 0.85­0.96). Hubo una buena concordancia entre los ensayos de PCR ITS1 y SCO 5995: E. acervulina (r = 0.92), E. maxima (r = 0.79) y E. tenella (r = 0.93). Sin embargo, solo el análisis ITS1 PCR corroboró los recuentos de micro-ooquistes de ADN de ooquistes de Eimeria recuperados de pollos de engorde infectados con Eimeria enviados a un laboratorio de diagnóstico avícola. Estos hallazgos sugieren que los métodos de ITS1 PCR o SCO PCR pueden validar los recuentos tradicionales de micro-ooquistes utilizados para cuantificar la infección por Eimeria en pollos. Estudios adicionales pueden proporcionar un método para estimar la abundancia relativa de cada especie de Eimeria en una infección natural.

Cloning and expression of a cDNA coding for Eimeria acervulina 25 kDa protein associated with oocyst and sporocyst walls.

The purpose of this study was to characterize a gene named EAH 00033530 identified by RNAseq analysis of sporulating Eimeria acervulina oocysts and its encoded protein. Quantitative RT-PCR analysis revealed peak expression of EAH 00033530 mRNA early (3-6 h) in sporulation followed by downregulation at 12-24 h. The gene for EAH 00033530 was expressed in Escherichia coli as a 70 kDa polyHis fusion protein (rEAH 00033530). Antisera prepared against rEAH 00033530 protein identified in immunoblotting a native 25 kDa E. acervulina protein (Ea25) that was present in oocyst-sporocyst extracts after treatment with the reducing agent DTT. Immunofluorescence staining using anti-rEa25 localized the protein to both E. acervulina oocyst and sporocyst walls, but not to sporozoites. The protein may be produced during in vivo oocyst development because immunostaining of duodenal tissue from E. acervulina-infected chickens revealed oocyst wall expression. As observed by ELISA, rEa25 protein appears to elicit a humoral immune response in chickens infected with non-irradiated or radiation-attenuated E. acervulina oocysts.

Gene expression during excystation of Cryptosporidium parvum oocysts.

The present study describes transcription of mRNA from genes encoding metabolic or structural proteins during excystation of Cryptosporidium parvum oocysts. RNA was harvested from C. parvum oocysts before excystation, and at 5, 10, and 15 min during excystation. Subtractive cDNA libraries were prepared by using mRNA from non-excysted C. parvum oocysts to "subtract out" mRNA from excysting oocysts. The "subtracted" cDNA was used to prepare libraries enriched for transcripts possibly involved in excystation. From these libraries, over 1,000 expressed sequence tags (ESTs) were analyzed by DNA sequencing followed by BLAST-N and BLAST-X analysis. While several gene products involved in cell metabolism and cell signaling were consistently recovered, transcription levels, as reflected by the relative number of cDNA sequences (19.2% total), were highly up-regulated in genes coding for structural proteins such as Cp2, CpTSP, CpHC10, and CpSAg. Moreover, of the greater than 1,000 clones analyzed, a high percentage (12.3%) of ESTs detected in excysting oocysts were for hypothetical C. parvum proteins (CpHyP), whose functions are presently unknown.

Dynamically expressed genes provide candidate viability biomarkers in a model coccidian.

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.