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The purpose of this study was to compare micro-oocyst counts ofiEimeria/ito PCR analysis of intestinal DNA from smears of duodenum, jejunum/ileum, and cecum of chickens infected withiEimeria acervulina/i,iEimeria maxima/i, oriEimeria tenella/ioocysts. Broiler chicks were infected in triplicate with various doses ofiE. acervulina/i,iE. maxima/i, oriE. tenella/ioocysts 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 eachiEimeria/ispecies. A strong correlation was found between mean micro-oocyst counts and both PCR assays foriE. acervulina/i(ir/i= 0.78-0.94),iE. maxima/i(ir/i= 0.79-0.91), andiE. tenella/i(ir/i= 0.85-0.96). There was good agreement between ITS1 and SCO 5995 PCR assays:iE. acervulina/i(ir/i= 0.92),iE. maxima/i(ir/i= 0.79), andiE. tenella/i(ir/i= 0.93). However, only ITS1 PCR analysis corroborated micro-oocyst counts ofiEimeria/ioocyst DNA recovered fromiEimeria/i-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 quantifyingiEimeria/iinfection in chickens. Additional studies may provide a method for estimating the relative abundance of eachiEimeria/ispecies in a natural infection.Reacción en cadena de la polimerasa dirigida al gene ITS1 rDNA deiEimeria/io a un ortólogo de copia única corrobora el análisis estándar de microquistes del tejido intestinal de pollos infectados coniE. acervulina/i,iE. maxima/ioiE. tenella/i. El propósito de este estudio fue comparar los recuentos de micro-ooquistes deiEimeria/icon el análisis PCR del ADN intestinal de frotis de duodeno, yeyuno/íleon y ciego de pollos infectados con ooquistes deiEimeria acervulina/i,iEimeria maxima/ioiEimeria tenella/i. Los pollos de engorde se infectaron por triplicado con varias dosis de ooquistes deiE. acervulina/i,iE. maxima/ioiE. tenella/iy 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 deiEimeria/i. Se encontró una fuerte correlación entre el recuento medio de micro-ooquistes y ambos ensayos de PCR paraiE. acervulina/i(ir/i= 0.78–0.94),iE. maxima/i(r = 0.79–0.91) yiE. tenella/i(r = 0.85–0.96). Hubo una buena concordancia entre los ensayos de PCR ITS1 y SCO 5995:iE. acervulina/i(ir/i= 0.92),iE. maxima/i(ir/i= 0.79) yiE. tenella/i(ir/i= 0.93). Sin embargo, solo el análisis ITS1 PCR corroboró los recuentos de micro-ooquistes de ADN de ooquistes deiEimeria/irecuperados de pollos de engorde infectados coniEimeria/ienviados 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 poriEimeria/ien pollos. Estudios adicionales pueden proporcionar un método para estimar la abundancia relativa de cada especie deiEimeria/ien una infección natural.

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.

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.

The intestinal disease coccidiosis, caused by parasiticEfectos de

The purpose of this study was twofold-first, to determine whether analysis of bacterial 16S ribosomal RNA (rRNA) in poultry litter corroborated standard

The purpose of this study was to evaluate the protective efficacy of a vaccine consisting of recombinant Neospora caninum-cyclophilin (NcCyP) and -profilin (NcPro) in sheep. At 42 d and 21 d prior to mating, adult Dorset ewes were immunized with the rNcCyP-rNcPro vaccine (Group 1) or co-purifying non-recombinant (NR) control vaccine (Group 2). At 90 days post-mating, all immunized ewes and were challenged by intravenous injection with 106Nesopora caninum Illinois tachyzoites (NcTZ). Significant protection (P < 0.05) was observed in Group 1 with 9 out of 13 ewes giving birth to live-born lambs (69.2%), whereas all Group 2 ewes aborted (6/6). Neospora caninum was detected by PCR in both fetal and placental tissues from all Group 2 aborting ewes and in the placental tissues of Group 1 aborting ewes. In contrast, tissues and placentas of Group 1 live-born lambs were Neospora DNA-negative. Immunoreactive Neospora antigens were demonstrated in placentas associated with abortions, but not in tissues of aborted fetuses or those of the live-born lambs and their associated placentas. Anti-NcCyP and anti-NcPro titers were high in sera from Group 1 ewes and were further boosted by challenge infection, resulting in long-lasting (≥14.5 mos.) elevated titers. Lambs born to Group 1 ewes also had high NcCyP and NcPro titers in pre-colostrum sera. Immunofluorescence staining (IFA) of NcTZ with Group 1 post-immunization sera revealed both surface and internal TZ staining, a pattern consistent with that observed with rabbit sera to rNcCyP or rNcPro. Infection of NR-vaccinated ewes produced high but transient anti-NcCyP and anti-NcPro Ab titers. The results indicate that the NcCyP-NcPro vaccine elicited strong anti-N. caninum responses and conferred significant protection against abortion and transplacental transmission of N. caninum TZ in sheep.

Butyrate is a feed additive that has been shown to have antibacterial properties and improve gut health in broilers. Here, we examined the performance and gene expression changes in the ileum of tributyrin–supplemented broilers infected with coccidia. Ninety-six, Ross 708 broilers were fed either a control corn–soybean–based diet (−BE) or a diet supplemented with 0.25% (w/w) tributyrin (+BE). Birds were further divided into groups that were inoculated with Eimeria maxima oocysts (EM) or sham-inoculated (C) on day 21 posthatch. At 7 d postinfection (7 d PI), the peak of pathology in E. maxima infection, tributyrin-supplemented birds had significantly improved feed conversion ratios (FCR, P < 0.05) and body weight gain (BWG, P < 0.05) compared with -BE-infected birds, despite both groups having similar feed intake (FI, P > 0.05). However, at 10 d post-infection (10 d PI) no significant effects of feed type or infection were observed. Gene expression in the ileum was examined for insights into possible effects of infection and tributyrin supplementation on genes encoding proteins related to immunity, digestion, and gut barrier integrity. Among immune-related genes examined, IL-1B and LEAP2 were only significantly affected at 7 d PI. Transcription of genes related to digestion (APN, MCT1, FABP2, and MUC2) were primarily influenced by infection at 7 d PI and tributyrin supplementation (FABP2 and MUC2) at 10 d PI. With exception of ZO1, tight junction genes were affected by either infection or feed type at 7 d PI. At 10 d PI, only CLDN1 was not affected by either infection or feed type. Overall tributyrin shows promise as a supplement to improve performance during coccidiosis in broiler chickens; however, its effect on gene expression and mode of action requires further research.

Made available in DSpace on 2019-10-04T12:35:30Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-01-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) USDA-ARS CRIS Project Sensitive detection of Cryptosporidium oocysts is important because the protozoan can cause clinical infection in humans at extremely low numbers. In the present study, 1.5 x 10(2), 1.0 x 10(3), or 1.0 x 10(4)C. parvum oocysts were spiked into 101 of source or finished water in triplicate followed by recovery using Envirochek HV sampling capsules. One subsample of the recovered oocysts was analyzed by commercial immuno fluorescence assay (IFA), while a second subsample was subjected to DNA-RNA extraction, followed by RT-PCR using primers directed to the gene encoding Cryspovirus capsid. IFA analysis of Envirochek filter eluates of finished water detected oocysts at all 3 C. parvum oocyst doses, but only at the 1.0 x 10(3) and 1.0 x 10(4) doses in source water. Cryspovirus RT-PCR appeared to offer greater sensitivity than IFA because C. parvum oocysts were detected using this molecular technique in both source and finished water concentrates at all 3 spiking levels. A linear relationship was observed between log oocysts spiking dose and the relative intensity of the Cryspovirus RT-PCR signal for finished water, but not for source water. These data indicate that Cryspovirus RT-PCR is a sensitive method for detecting C. parvum oocysts in source and finished water. Univ Estadual Paulista, Coll Vet Med, Dept Clin Surg & Anim Reprod, Clovis Pestana 793, BR-16050680 Sao Paulo, Brazil ARS, Environm Microbial & Food Safety Lab, USDA, Beltsville, MD 20705 USA Univ Estadual Paulista, Coll Vet Med, Dept Clin Surg & Anim Reprod, Clovis Pestana 793, BR-16050680 Sao Paulo, Brazil FAPESP: 2015/07147-2 USDA-ARS CRIS Project: 8042-32000-100-00-D

A programmed self-destructive Salmonella vaccine delivery system was developed to facilitate efficient colonization in host tissues that allows release of the bacterial cell contents after lysis to stimulate mucosal, systemic, and cellular immunities against a diversity of pathogens. Adoption and modification of these technological improvements could form part of an integrated strategy for cost-effective control and prevention of infectious diseases, including those caused by parasitic pathogens. Avian coccidiosis is a common poultry disease caused by Eimeria. Coccidiosis has been controlled by medicating feed with anticoccidial drugs or administering vaccines containing low doses of virulent or attenuated Eimeria oocysts. Problems of drug resistance and nonuniform administration of these Eimeria resulting in variable immunity are prompting efforts to develop recombinant Eimeria vaccines. In this study, we designed, constructed, and evaluated a self-destructing recombinant attenuated Salmonella vaccine (RASV) lysis strain synthesizing the Eimeria tenella SO7 antigen. We showed that the RASV lysis strain χ11791(pYA5293) with a ΔsifA mutation enabling escape from the Salmonella-containing vesicle (or endosome) successfully colonized chicken lymphoid tissues and induced strong mucosal and cell-mediated immunities, which are critically important for protection against Eimeria challenge. The results from animal clinical trials show that this vaccine strain significantly increased food conversion efficiency and protection against weight gain depression after challenge with 105E. tenella oocysts with concomitant decreased oocyst output. More importantly, the programmed regulated lysis feature designed into this RASV strain promotes bacterial self-clearance from the host, lessening persistence of vaccine strains in vivo and survival if excreted, which is a critically important advantage in a vaccine for livestock animals. Our approach should provide a safe, cost-effective, and efficacious vaccine to control coccidiosis upon addition of additional protective Eimeria antigens. These improved RASVs can also be modified for use to control other parasitic diseases infecting other animal species.

Fayoumi chickens are believed to be more disease resistant compared to commercial broiler chickens. The objective of this study was to compare mRNA expression of intestinal nutrient transporters, digestive enzymes, and host defense peptides (HDP) between Eimeria maxima-challenged Fayoumi and Ross broiler chickens. At 21 d of age, Ross broilers and Fayoumi lines M5.1 and M15.2 were challenged with 1,000 E. maxima oocysts. Control birds were not challenged. Duodenum, jejunum, and ileum were sampled (n = 6) at 7 d post challenge. Gene expression was analyzed using relative quantification PCR. Data were analyzed by ANOVA and significance level was set at P< 0.05. There was numerical, but not statistically significant, differential weight gain depression for Ross (15%) and Fayoumi lines M5.1 (21%) and M15.2 (22%) and significant line-specific changes in gene expression. For nutrient transporters, there was downregulation of mRNA for the brush border membrane, amino acid transporters b(0,+) AT/rBAT, BoAT, and EAAT3 in different segments of the small intes-tine of Ross and both lines of Fayoumi chickens, indicating that E. maxima challenge likely caused a decrease in nutrient uptake. For HDP, there was downregulation of avian beta defensin (AvBD) 1, 6, 10, 12, and 13 mRNA in the jejunum of the 2 Fayoumi lines, but no change in the Ross broilers. In the duodenum, there was upregulation of AvBD10 mRNA in the Ross and both Fayoumi lines and additionally upregulation of AvBD11, 12, and 13 mRNA in only Fayoumi line M15.2. Liver expressed antimicrobial peptide 2 (LEAP2) mRNA was downregulated in the duodenum and jejunum of Ross and Fayoumi line M5.1 but not in Fayoumi line M15.2. The homeostatic, non-challenged levels of AvBD mRNA were greater in Fayoumi line M15.2 than Ross and Fayoumi line M5.1 in the duodenum and ileum. This study demonstrates tissueand genetic line-specific transcriptional responses to E. maxima, highlights novel potential candidate genes for response to coccidiosis, and confirms a role for several previously reported genes in response to coccidiosis. College of Agriculture and Life Sciences Pratt Endowment at Virginia Tech; Virginia Agricultural Experiment Station; Hatch Program of the National Institute of Food and Agriculture, U.S. Department of Agriculture; Animal Health Formula Funds; Hatch project, Iowa State University [5458] This work was funded in part by the College of Agriculture and Life Sciences Pratt Endowment at Virginia Tech (SS), the Virginia Agricultural Experiment Station and the Hatch Program of the National Institute of Food and Agriculture, U.S. Department of Agriculture (EAW), and Animal Health Formula Funds and Hatch project number 5458, Iowa State University (SJL). Public domain – authored by a U.S. government employee

The purpose of this study was to optimize primary and nested polymerase chain reaction (PCR) assays for detecting the microsporidia Encephalitozoon intestinalis and Enterocytozoon bieneusi in fecal samples from dairy calves. PCR for these microsporidia were compared to immunofluorescence assays (IFA) based on commercially available monoclonal antibodies specific for outer wall proteins of Enc. intestinalis or Ent. bieneusi. Fecal samples were collected from 15 dairy calves and processed by molecular sieving followed by salt floatation to recover Enc. intestinalis and Ent. bieneusi spores. An aliquot of the final supernatant was applied to glass slides for IFA testing; another aliquot was extracted for total DNA using a QIAamp Stool Mini-Kit for primary and nested Enc. intestinalis- and Ent. bieneusi-specific PCR analysis. Internal standards were generated for both Enc. intestinalis and Ent. bieneusi PCR assays to control for false negative reactions due to the presence of inhibitors commonly found in fecal samples. Using the commercial MicrosporIFA (Waterborne, Inc.) as the gold standard, the optimized Enc. intestinalis PCR method provided 85.7% sensitivity and 100% specificity with a kappa value = 0.865. Likewise, using the commercial BienusiGlo IFA (Waterborne, Inc.) as the gold standard, the optimized Ent. bieneusi PCR method provided 83.3% sensitivity and 100% specificity with a kappa value = 0.857. Sequencing of amplicons from both PCR assays confirmed the presence of Enc. intestinalis or Ent. bieneusi. In conclusion, our optimized assays for recovering and detecting Enc. intestinalis or Ent. bieneusi in feces from dairy calves provides a valuable alternative to traditional IFA methods that require expertise to identify extremely small microsporidia spores (~ 2.0 µm). Our assays also improve upon existing molecular detection techniques for these microsporidia by incorporating an internal standard to control for false negative reactions.

Cryptosporidium spp. is a zoonotic intracellular protozoan and a significant cause of diarrhoea in humans and animals worldwide. This parasite can cause high morbidity in immunocompromised people and children in developing countries, livestock being the main reservoir. This study was aimed at performing preliminary tests on Swiss albino weaned mice (ICR) to evaluate the humoral immune response induced against peptides derived from Cryptosporidium parvum CP15 (15 kDa sporozoite surface antigen) and CSL (circumsporozoite-like antigen) proteins. Peptides were identified and characterised using bioinformatics tools and were chemically synthesised. The antibody response was determined and the neutralising effect of antibodies was measured in cell culture. Despite all peptides studied here were capable of stimulating antibody production, neutralising antibodies were detected for just two of the CP15-derived ones. Additional studies aimed at evaluating further the potential of such peptides as vaccine candidates are thus recommended. © 2018 Elsevier Ltd

The importance of birds in the biological cycle of Neospora caninum is not clear. We report unsuccessful Neospora infection in chickens (Gallus gallus domesticus) using two isolates of N. caninum. In experiment #1, 30 White Leghorn chickens were orally inoculated with viable N. caninum oocysts (NC-SP1 isolate, 200 oocysts per bird) via the crop at 21days of age. Groups of three birds were euthanised at intervals of 7days (a total of 9weeks) and one group was challenged with the same oocyst dose at 37daysp.i. and observed for 11weeks. Blood samples were collected weekly, and sera were tested using IFAT. Chicken tissues were collected for PCR, quantitative PCR and immunohistochemistry. Two dogs approximately 45days of age were fed with tissues from chickens euthanised at 138 and 159daysp.i. The results indicated that the chickens were resistant to neosporosis as revealed by failure to seroconvert, to detect parasite DNA or N. caninum antigen by immunohistochemistry in inoculated bird tissues, and by no oocyst excretion by the dogs fed avian tissues. Similar results were obtained in experiment #2, in which 34 1-week-old chickens were each s.c. inoculated with 100,000 tachyzoites of the NcWTDMn1 isolate of N. caninum. The chickens were euthanised on days 7, 15, 22, 28, 36 and 60p.i. At necropsy, all tissues and serum from each bird were collected. All chickens remained asymptomatic, and N. caninum antigen was not detected by immunohistochemistry. Seven chickens euthanised at day 60p.i. demonstrated low (1:25 dilution) levels of antibodies by using the Neospora agglutination test. Two 12-week-old dogs fed tissues pooled from 10 inoculated chickens euthanised at day 60p.i. did not excrete N. caninum oocysts. This investigation indicates that chickens are resistant to experimental infection by N. caninum.

Ostertagiosis remains an economically important parasitic disease in cattle in the temperate regions of the world. Repeated exposures to Ostertagia ostertagi in calves cause significant pathology in the abomasum but elicit little protective immunity. The larvae use the host’s gastric glands as a niche for development, where the parasite completes its parasitic stages, while in the gastric glands, the larvae must down-regulate the host inflammatory immune responses. Annexin (ANX) A1, commonly found in most eukaryotes, is heavily involved in controlling anti-inflammatory responses by binding receptors on leukocytes. We hypothesized, therefore, that parasite proteins of the ANX family may be involved in host-parasite interactions during ostertagiosis. BLASTN search with the bovine ANXA1 identified two families of Oos-ANX like proteins (Oos-ANXL), each of which was highly conserved at the genetic level and identical at the amino acid sequence level. Oos-ANXL-1 is encoded by two transcripts and Oos-ANXL-2 by 20 transcripts. The present study characterized one Oos-ANXL, representing the most abundant Oos-ANXL, which was further defined as Oost-ANXL-2.1. Oos-ANXL-2.1 with a coding sequence of 519 bp was PCR-amplified, cloned, and expressed. Oos-ANXL-2.1 was immunolocalized to both L3 and adult, but not L4. The staining appeared to be associated with the gut and hypodermis in L3, but it was specifically localized to the hypodermis in adult worms. Western blots detected three protein bands in parasite lysates using anti-recombinant Oos-ANXL-2.1 antibody. Integrated optical density for each of the 3 Oos-ANXL-2s or the total Oos-ANXL-2s detected by Western blots (P

The aim of the present study was to evaluate oocyst shedding in cats immunized by nasal route with T. gondii proteins ROP2. Twelve short hair cats (Felis catus) were divided in three groups G1, G2 and G3 (n=4). Animals from G1 received 100 μg of rROP2 proteins plus 20 μg of Quil-A, G2 received 100 μg of BSA plus 20 μg of Quil-A, and the G3 only saline solution (control group). All treatments were done by intranasal route at days 0, 21, 42, and 63. The challenge was performed in all groups on day 70 with ≅ 800 tissue cysts of ME-49 strain by oral route. Animals from G1 shed less oocysts (86.7%) than control groups. ELISA was used to detect anti-rROP2 IgG and IgA, however, there were no correlation between number of oocyst shedding by either IgG or IgA antibody levels. In the present work, in spite of lesser oocysts production in immunized group than control groups, it was not possible to associate the use of rROP2 via nostrils with protection against oocyst shedding. For the future, the use of either other recombinant proteins or DNA vaccine, in combination with rROP2 could be tested to try improving the efficacy of this kind of vaccine.

Eimeria maxima is one of the most pathogenic species of avian coccidia, yet it is unknown why different E. maxima strains differ in the pathogenic effects they cause in chickens. The purpose of this study was to determine if a more pathogenic E. maxima strain (APU1) was also more fecund than a less pathogenic E. maxima strain (APU2). At identical doses, E. maxima APU1 always produces greater intestinal lesions and lower weight gain compared to E. maxima APU2. Using a dose response study, median and mean intestinal lesion scores in E. maxima APU1-infected chickens were greater by a score of 1-1.5 compared to chickens infected with E. maxima APU2. Likewise, weight gain depression in E. maxima APU1-infected chickens was 20-25% greater (equivalent to 110-130g body weight) than in E. maxima APU2-infected chickens. In order to understand the underlying cause of these observed clinical effects, 120 broiler chicks (5 oocyst levels, 6 replicates/level) were inoculated with various doses of E. maxima APU1 or APU2 oocysts. The dynamics of oocyst shedding was investigated by collecting fecal material every 12h from 114 to 210h post-inoculation (p.i.) and every 24h thereafter from 210 to 306h, and then processed for measuring E. maxima oocyst output. Oocysts were first observed at 138h p.i., and time of peak oocyst production was nearly identical for both E. maxima APU1 and APU2 around 150-162h. Total oocyst production was 1.1-2.6 fold higher at all dose levels for E. maxima APU1 compared to E. maxima APU2, being significantly higher (P0.05) at the log 1.5 dose level. Other groups of chickens were infected with higher doses of E. maxima APU1 or APU2 oocysts, and intestinal lesions were assessed by histology at 72, 96, 120, and 144h p.i. Although schizonts, gamonts, and oocysts were observed at expected time-points, no obvious differences were noted in lesions induced by the two E. maxima strains. This study showed that the greater fecundity of E. maxima APU1 compared to E. maxima APU2 explains in part the observed differences in pathogenicity of the two E. maxima strains, but that other factors may contribute to differences in observed clinical effects.

Release of sporozoites from Eimeria oocysts/sporocysts is an essential step in the intracellular development of the parasite in its host. Little is known about this process except that elevated temperature (∼ 40 °C) plus trypsin and bile salts are required for sporozoite to escape from sporocysts. In this study, it was found that adding a reducing agent, either dithiothreitol (DTT) or Tris(2-carboxyethyl)phosphine hydrochloride (TCEP), increased the lifespan of sporozoites released from Eimeria maxima. While the addition of DTT or TCEP affected the apparent molecular weight of trypsin, it did not interfere with excystation of E. maxima, but rather had a positive effect on the number of viable sporozoites present after release. This effect was time-dependent in that the number of intact sporozoites at 15 and 30 min after excystation was similar between untreated and DTT- or TCEP-treated sporocysts. However, by 45 – 60 min, virtually no sporozoites were observed in excystation fluid not containing DTT or TCEP. Of interest is that this effect appeared to be Eimeria species-dependent. Eimeria acervulina and E. tenella sporozoites remained viable for at least 60 min after excystation in the absence of DTT or TCEP. The effect of DTT and TCEP on chymotrypsin was also studied with all 3 Eimeria species because there is some evidence that chymotrypsin is an effective excystation enzyme. Indeed, E. maxima sporozoites excysting from sporocysts with chymotrypsin in the presence of DTT or TCEP remained viable for at least 60 min after release, unlike excystation done in the absence of these reducing agents. Chymotrypsin was capable of excysting E. acervulina in the presence or absence of DTT or TCEP. Of interest, is that chymotrypsin was ineffective in the excystation of E. tenella. These findings suggest that trypsin and chymotrypsin have differential effects on sporozoite excystation and that reducing agents may alter sites on the enzyme that affect sporozoite viability, but not release from sporocysts.

The purpose of the present study was to determine whether a correlation existed between chick mortality and the presence of Clostridium perfringens alpha-toxin and NetB-toxin genes (cpa and netB) in C. perfringens recovered from litter in commercial broiler houses. Because coccidiosis predisposes chickens to necrotic enteritis, the concentration of Eimeria oocysts in these samples was measured, and the numbers were used in similar correlation analyses. Litter samples were collected at 0, 2, and 4 wk growout from six broiler farms (18 houses total) during an anticoccidial drug (ACD) control program and from nine broiler farms (23 houses total) during an Eimeria vaccine (VAC) control program. Of these, litter samples were collected from five farms during both ACD and VAC programs. The litter samples were processed for Eimeria oocyst and C. perfringens spore enumerations by standard parasitologic and microbiologic techniques. DNA was also extracted for C. perfringens DNA for PCR detection of genes coding for alpha- and NetB-toxin. A general trend during the ACD programs was a transient decrease in both Eimeria maxima and non-E. maxima (Eamipt) numbers at 2 wk growout. The pattern was slightly different during VAC with E. maxima and Eamipt levels increasing over time. Average concentrations of C. perfringens in litter were highest at 2 wk (∼105-106 spores/g) during ACD and at placement during VAC (∼105-106 spores/g). During the ACD program, a strong correlation was observed between 0 and 3-wk chick mortality and the presence at placement (0 wk) of netB (r = 0.42-0.48) or cpa (r = 0.55-0.67). A very strong correlation was observed in 0-5-wk chick mortality and the presence of netB at 4 wk growout (0.73-0.95). During a VAC program, a strong correlation was only observed between the presence of netB at placement and 0-1-wk chick mortality (r = 0.67).

Cryptosporidium parvum represents a considerable health risk to humans and animals because the parasite has a low infectious dose and is usually present at low numbers in environmental samples, which makes detection problematic. The purpose of this study was to evaluate Cryspovirus as a target for sensitive detection of C. parvum in clinical samples. Semi-quantitative RT-PCR (sqRT-PCR) and quantitative RT-PCR (qRT-PCR) directed to Cryspovirus sequences could detect less than 5 Cryptosporidium oocysts in RNA extracted from C. parvum-containing calf feces. Of interest was that a similar level of sensitivity was observed using RNA present in DNA extracts of the same C. parvum fecal samples. There was a strong correlation between both the sqRT-PCR and qRT-PCR product and number of C. parvum oocysts. Analysis of DNA extracted from a similar number of oocysts using PCR targeting the Cryptosporidium SSU rDNA gene sequence found that nested PCR was necessary to obtain a detectable PCR signal. The availability of DNA allowed for Cryptosporidium genotyping based on SSU rDNA sequencing as well as C. parvum subtyping through GP60 sequencing. By using DNA that contains viral RNA, the assay avoids two separate extractions — one for RNA and one for DNA. This two-step assay, first to detect Cryptosporidium by Cryspovirus-specific RT-PCR followed by nested SSU rDNA PCR for Cryptosporidium genotyping may represent an important tool for identifying the parasite in clinical samples. Keywords: Cryptosporidium, Cryspovirus, Detection, RT-PCR

The present study represents the first description of ionophore resistance in recovered from commercial Algerian (Jijel-Algeria) broiler farms. Microscopy and intervening transcribed sequence 1 PCR (ITS1 PCR) revealed only 2 Eimeria species present in litter from these farms- namely Eimeria acervulina and Eimeria maxima. A pool of these isolates were evaluated in broiler chickens (Cobb 500) for sensitivity to 5 anticoccidial compounds-diclazuril (1ppm), lasalocid (125ppm), monensin (125ppm), narasin (70ppm) and salinomycin (60ppm). As indicated by anticoccidial sensitivity profiles based on lesion scores and anticoccidial index (ACI), complete resistance to monensin and narasin, partial resistance to salinomycin and lasalocid, and complete sensitivity to diclazuril was observed. While lack of sensitivity to monensin is not surprising given its use for years as the sole anticoccidial compound, the resistance to monoether (narasin) and polyether (lasalocid) ionophores suggests that cross-resistance has developed in a segment of the Eimeria population. The fairly uniform Eimeria species composition among all poultry farms suggests that E. acervulina and E. maxima more rapidly develop resistance to ionophore drugs.

Eimeria, protozoan parasites from the phylum Apicomplexa, can cause the enteric disease coccidiosis in all farmed animals. Coccidiosis is commonly considered to be most significant in poultry; due in part to the vast number of chickens produced in the World each year, their short generation time, and the narrow profit margins associated with their production. Control of Eimeria has long been dominated by routine chemoprophylaxis, but has been supplemented or replaced by live parasite vaccination in a minority of production sectors. However, public and legislative demands for reduced drug use in food production is now driving dramatic change, replacing reliance on relatively indiscriminate anticoccidial drugs with vaccines that are Eimeria species-, and in some examples, strain-specific. Unfortunately, the consequences of deleterious selection on Eimeria population structure and genome evolution incurred by exposure to anticoccidial drugs or vaccines are unclear. Genome sequence assemblies were published in 2014 for all seven Eimeria species that infect chickens, stimulating the first population genetics studies for these economically important parasites. Here, we review current knowledge of eimerian genomes and highlight challenges posed by the discovery of new, genetically cryptic Eimeria operational taxonomic units (OTUs) circulating in chicken populations. As sequencing technologies evolve understanding of eimerian genomes will improve, with notable utility for studies of Eimeria biology, diversity and opportunities for control.

The purpose of this study was to determine if Eimeria oocysts recovered from litter at the time of chick placement in commercial broiler houses contained oocysts that were infectious for chickens. Over 100 litter samples were collected from 30 poultry farms representing a total of 60 different broiler houses with 9 houses sampled more than once over 1.5 yr. The samples were collected just before the placement of newly hatched chicks and after an anticoccidial drug (ACD) or Eimeria vaccine (VAC) program, and processed for counting oocysts followed by Eimeria species determination using ITS1 PCR. Broiler chicks were inoculated with recovered Eimeria oocysts to determine if the litter oocysts were viable and capable of causing patent infection. At placement, E. maxima (Emax) oocysts were detected in 70 of 75 houses after ACD program and 46 of 47 houses after VAC program. Eimeria acervulina, E. praecox, and/or E. tenella (Eapt) were detected in 75 of 75 houses after ACD program and 47 of 47 houses after VAC program. Viability testing revealed that 33.0% of broiler houses contained viable Emax oocysts, while 46.9% contained viable Eapt oocysts. During VAC programs, the concentration of Emax oocysts at placement and the total number of Emax oocysts shed by chickens in viability studies showed a very strong correlation (r = 0.83). Likewise, during ACD programs, the concentration of Eapt oocysts at placement and the total number of Eapt oocysts shed by chickens in the viability study showed a strong correlation (r = 0.62). In general, Eimeria oocyst levels at placement and number of viable oocysts shed by chickens in the viability study were similar among houses on the same farm. However, the number of Eimeria oocysts shed in the viability studies was considerably less than expected based on the number of oocysts given. These data suggest that nearly 100% of all poultry houses contain Emax and Eapt oocysts at placement with 30 to 50% of the houses containing viable Eimeria oocysts, thus possibly representing a source of the protozoa to newly hatched chicks.

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 protozoan Neospora caninum is transmitted between domestic and wildlife species. Urbanized environments and deer density may facilitate this transmission and play a critical role in the spillover of N. caninum from domestic animals to wildlife. White-tailed deer (Odocoileus virginianus; WTD) are an important intermediate host for maintaining the sylvatic cycle of N. caninum in the USA. Here, we assayed serum samples from 444 WTD from a nature reservation across a suburban to urban gradient in Ohio, USA. Antibodies to N. caninum were found by using a recombinant NcGRA6 ELISA in 23.6% (105/444). Significant risk factors for seropositivity were age class and urbanization. Deer from urbanized environments were at greater odds of being seropositive (89/323, 27.6%) than those from suburban habitats (16/121, 13.2%), and this difference persisted when adjusting for age and sex. Age was also a significant risk factor with adults at greater odds to be seropositive than fawns and yearlings. We speculate the main route of exposure in WTD is ingestion of N. caninum oocysts from contaminated environments and urbanized habitats facilitate this exposure.

The purpose of this study was to determine if conjugating a recombinant Eimeria maxima protein, namely EmaxIMP1, into 20 nm polystyrene nanoparticles (NP) could improve the level of protective immunity against E. maxima challenge infection. Recombinant EmaxIMP1 was expressed in Escherichia coli as a poly-His fusion protein, purified by NiNTA chromatography, and conjugated to 20 nm polystyrene NP (NP-EmaxIMP1). NP-EMaxIMP1 or control non-recombinant (NP-NR) protein were delivered per os to newly-hatched broiler chicks with subsequent booster immunizations at 3 and 21 days of age. In battery cage studies (n = 4), chickens immunized with NP-EMaxIMP1 displayed complete protection as measured by weight gain (WG) against E. maxima challenge compared to chickens immunized with NP-NR. WG in the NP-EMaxIMP1-immunized groups was identical to WG in chickens that were not infected with E. maxima infected chickens. In floor pen studies (n = 2), chickens immunized with NP-EMaxIMP1 displayed partial protection as measured by WG against E. maxima challenge compared to chickens immunized with NP-NR. In order to understand the basis for immune stimulation, newly-hatched chicks were inoculated per os with NP-EMaxIMP1 or NP-NR protein, and the small intestine, bursa, and spleen, were examined for NP localization at 1 h and 6 h post-inoculation. Within 1 h, both NP-EMaxIMP1 and NP-NR were observed in all 3 tissues. An increase was observed in the level of NP-EmaxIMP1 and NP-NR in all tissues at 6 h post-inoculation. These data indicate that 20 nm NP-EmaxIMP1 or NP-NR reached deeper tissues within hours of oral inoculation and elicited complete to partial immunity against E. maxima challenge infection.

The purpose of this study was to determine if Eimeria oocyst concentrations and species composition in commercial broiler house litter changed during different cycles of anticoccidial drug (ACD) or live Eimeria oocyst vaccine (VAC) control programs and if there was a correlation between Eimeria oocyst levels and broiler performance. Litter samples were collected from a total of 15 different broiler farms encompassing a total of 45 individual houses during at least one complete grow-out cycle over a 21-mo period. Of these 15 broiler farms, three were followed for the entire 21-mo period spanning three ACD and four VAC cycles. Samples were collected at 2, 4, and 7-8 wk of grow-out corresponding to starter, grower, and withdraw periods of the ACD cycle. On a number of occasions, litter samples were obtained just prior to chick placement. Eimeria oocysts were isolated from all samples, counted by microscopy, and extracted for DNA to identify Eimeria species by ITS1 PCR. In general, Eimeria oocyst concentration in litter reached peak levels at 2-4 wk of grow-out regardless of coccidiosis control measure being used. However, peak oocyst numbers were sometimes delayed until 7-8 wk, indicating some level of Eimeria spp. drug resistance or incomplete vaccine coverage. Eimeria maxima , Eimeria acervulina , Eimeria praecox, and Eimeria tenella were generally present in all samples, and no difference in the species composition was noted between houses on a particular farm. While Eimeria species composition was similar among houses, Eimeria spp. oocyst levels exhibited sporadic peaks in one house of a given location's houses. Of particular interest was the observed correlation between E. maxima oocyst abundance and chick mortality. However, no correlation was observed in E. maxima oocyst levels, and the performance parameters adjusted feed conversion ratio and average daily weight gain. This study showed that understanding the dynamics of Eimeria spp. oocyst levels and species composition in litter during ACD or VAC programs may provide insight into the effectiveness of coccidiosis control measures in commercial broiler production.

Standard methods of determining the ionophore sensitivity of Eimeria rely on infecting chickens with an isolate or a mixture of Eimeria spp. oocysts in the presence of different anti-coccidial drugs. The purpose of this study was to develop a rapid in vitro method for assessing salinomycin and monensin sensitivity in Eimeria tenella. Cultures of MDBK cells were grown to 85% confluency, and then inoculated with excysted E. tenella laboratory strain (APU-1) sporozoites in the presence of different concentrations of salinomycin or monensin. At various timepoints, the monolayers were fixed for counting intraceullar sporozoites, or were subjected to DNA extraction, followed by molecular analysis using quantitative (qPCR) or semi-quantitative PCR (sqPCR). Preliminary experiments showed that 24 h was the optimum time for harvesting the E. tenella-infected cell cultures. The average number of E. tenella sporozoites relative to untreated controls displayed a linear decrease between 0.3 and 33.0 μg/ml salinomycin and between 0.3 and 3.3 μg/ml monensin. A similar pattern was observed in the relative amount of E. tenella DNA as measured by sqPCR. A linear decrease in the relative amount of E. tenella DNA was observed over the entire range of salinomycin and monensin concentrations as measured by qPCR possibly reflecting the greater sensitivity of this assay. Comparison of sporozoite counting, sqPCR, and qPCR signals using a criterion of 50% inhibition in sporozoite numbers or level of PCR amplification product showed good agreement between the three assays. E. tenella field isolates (FS-1 and FS-2) displaying resistance to salinomycin and monensin were evaluated in the in vitro assay using qPCR and sqPCR. Compared to E. tenella APU-1, the E. tenella FS-1 and FS-2 isolates showed higher levels of E. tenella DNA at 24 h by both qPCR and sqPCR. This in vitro assay represents a significant advance in developing rapid, cost-effective methods for assessing ionophore sensitivity in E. tenella.

Previous studies comparing the genome sequences of Cryptosporidium parvum with Cryptosporidium hominis identified a number of highly divergent genes that might reflect positive selection for host specificity. In the present study, the C. parvum DNA sequence cgd8-5370, which encodes a protein whose amino acid sequence differs appreciably from its homologue in C. hominis , was cloned by PCR and expressed as a recombinant protein in Escherichia coli . Antisera raised against the recombinant cgd8-5370 antigen strongly recognized a unique 33 kDa protein in immunoblots from reducing and non-reducing SDS-PAGE of native C. parvum protein. However, anti-Cp33 sera did not recognize the native 33 kDa homologue in C. hominis . In an immunofluorescence assay (IFA), anti-Cp33 serum recognized an antigen in the anterior end of air-dried C. parvum sporozoites but failed to bind at any sites in C. hominis sporozoites, indicating its specificity for C. parvum . IFA staining of live C. parvum sporozoites with anti-Cp33 serum failed to bind to the parasite, indicating that the CP33 antigen is not on the sporozoite surface, which is consistent with topology predictions based on the encoded amino acid sequence. RT-PCR analysis of cgd8-5370 mRNA before or during C. parvum oocyst excystation revealed transcripts only in excysting sporozoites. Thus, Cp33 represents one of a small number of proteins shown to differentiate C. parvum from C. hominis sporozoites and oocysts.

Neospora caninum is a common cause of abortion in cattle worldwide. Canids, including the dog and the dingo (Canis familiaris), the coyote (Canis latrans), and the gray wolf (Canis lupus) are its definitive hosts that can excrete environmentally resistant oocysts in the environment, but also can act as intermediate hosts, harboring tissue stages of the parasite. In an attempt to isolate viable N. caninum from tissues of naturally infected wolves, brain and heart tissue from 109 wolves from Minnesota were bioassayed in mice. Viable N. caninum (NcWolfMn1, NcWolfMn2) was isolated from the brains of two wolves by bioassays in interferon gamma gene knockout mice. DNA obtained from culture-derived N. caninum tachyzoites of the two isolates were analyzed by N. caninum-specific Nc5 polymerase chain reaction and confirmed diagnosis. This is the first report of isolation of N. caninum from tissues of any wild canid host.

Coccidiosis is a major contributor to economic losses in the poultry industry due to its detrimental effects on growth performance and nutrient utilization. We hypothesized that the combined effects of supplemental dietary Thr and purified fiber may modulate the intestinal environment and positively affect intestinal immune responses and barrier function in broiler chicks infected with Eimeria maxima. A Thr-deficient basal diet (3.1 g of Thr/kg of diet) was supplemented with 70 g/kg of silica sand (control) or high-methoxy pectin and 1 of 2 concentrations of Thr (1.8 or 5.3 g/kg of diet; 4 diets total), and fed to chicks from hatch to d 16 posthatch. On d 10 posthatch, chicks received 0.5 mL of distilled water or an acute dose of Eimeria maxima (1.5 × 10(3) sporulated oocytes) with 6 replicate pens of 6 chicks per each of 8 treatment combinations (4 diets and 2 inoculation states). Body weight gain, feed intake, and G:F increased (P < 0.01) with addition of 5.3 g of Thr/kg of diet. Eimeria maxima schizonts were present only in intestinal tissue sampled from infected birds (P < 0.01). Weights of cecal digesta were highest (P < 0.01) in pectin-fed birds, and ceca with the heaviest weights also had the highest concentrations of total short-chain fatty acids. Expression of interleukin-12 in ileal mucosa was highest (P < 0.01) in infected birds receiving the control diet with 5.3 g of supplemental Thr/kg. In cecal tonsils, interferon-γ expression was highest in infected birds receiving the control diet (fiber × infection, P < 0.05); interferon-γ expression was lowest in infected birds fed the high Thr diet (Thr × infection, P < 0.05). There were no differences due to infection or Thr supplementation for cytokine expression in birds fed pectin-containing treatments. Overall, we conclude that although pectin has some protective function against coccidiosis, Thr supplementation had the greatest effect on intestinal immune response and maintenance of near normal growth in young broiler chicks infected with E. maxima.

Avian coccidiosis is caused by the intracellular protozoan Eimeria, which produces intestinal lesions leading to weight gain depression. Current control methods include vaccination and anticoccidial drugs. An alternative approach involves modulating the immune system. The objective of this study was to profile the expression of host defense peptides such as avian beta-defensins (AvBDs) and liver expressed antimicrobial peptide 2 (LEAP2), which are part of the innate immune system. The mRNA expression of AvBD family members 1, 6, 8, 10, 11, 12, and 13 and LEAP2 was examined in chickens challenged with either E. acervulina, E. maxima, or E. tenella. The duodenum, jejunum, ileum, and ceca were collected 7 d post challenge. In study 1, E. acervulina challenge resulted in down-regulation of AvBD1, AvBD6, AvBD10, AvBD11, AvBD12, and AvBD13 in the duodenum. E. maxima challenge caused down-regulation of AvBD6, AvBD10, and AvBD11 in the duodenum, down-regulation of AvBD10 in the jejunum, but up-regulation of AvBD8 and AvBD13 in the ceca. E. tenella challenge showed no change in AvBD expression in any tissue. In study 2, which involved challenge with only E. maxima, there was down-regulation of AvBD1 in the ileum, AvBD11 in the jejunum and ileum, and LEAP2 in all 3 segments of the small intestine. The expression of LEAP2 was further examined by in situ hybridization in the jejunum of chickens from study 2. LEAP2 mRNA was expressed similarly in the enterocytes lining the villi, but not in the crypts of control and Eimeria challenged chickens. The lengths of the villi in the Eimeria challenged chickens were less than those in the control chickens, which may in part account for the observed down-regulation of LEAP2 mRNA quantified by PCR. Overall, the AvBD response to Eimeria challenge was not consistent; whereas LEAP2 was consistently down-regulated, which suggests that LEAP2 plays an important role in modulating an Eimeria infection.