Exploiting digital droplet PCR and Next Generation Sequencing technologies to determine the relative abundance of individual Eimeria species in a DNA sample.

DNA-based diagnostic assays for detecting infections with Eimeria species have been limited to providing identification and presence/absence data for samples containing oocysts. Modern technologies that generate quantitative data, such as droplet digital PCR (ddPCR) and Next Generation Sequencing (NGS), utilize a relatively short amplicon size containing sufficient species-specific variation for reliable species level identification. Targeting the cytochrome c oxidase subunit III gene in the mitochondrial genome, we established protocols using these technologies to determine the relative abundance of the number of copies/µL of Eimeria species in a sample. Samples from chickens of known and unknown Eimeria species composition were analyzed to determine the suitability of these technologies as diagnostic assays. All technologies demonstrated robust capability of identifying and quantifying the Eimeria species in samples. The new quantitative assays described herein will produce invaluable detail of Eimeria species infections for an array of situations in commercial chicken production systems, enabling further characterization of the disease profile and allowing for the development or enhancement of new intervention strategies.

Restoration of anticoccidial sensitivity to a commercial broiler chicken facility in Canada.

Increasing resistance of Eimeria species to anticoccidial medications is an issue in the broiler chicken industry. Using drug-sensitive strains in live-coccidiosis vaccines has been shown to improve anticoccidial effectiveness in US-based broiler production. In Canada, litter is removed between flocks, which differ from the US industry practice. Thus, we investigated the use of drug-sensitive vaccine strains in a Canadian broiler production facility with suspected anticoccidial resistance. Weekly fecal samples were collected from flocks before, during, and after vaccine seeding to determine oocyst shedding patterns; following the vaccine seeding, OPG counts from similar aged birds were lower than flocks before live-coccidiosis vaccine use. Eimeria species isolates, collected before and after vaccine seeding, were used in 2 anticoccidial sensitivity tests to evaluate their susceptibility to commercially available anticoccidial medications; a low-dose challenge to define parasite replication, and a high-dose challenge to monitor broiler performance. In both experiments, isolates collected after seeding were more susceptible to almost every anticoccidial medication evaluated compared with the isolates collected before seeding. These results demonstrate an improvement in sensitivity to many anticoccidials after the use of live-coccidiosis vaccines at this facility. However, the regulated removal of litter at the end of each flock required under Canadian broiler chicken production management rules could limit the establishment of vaccine-strain Eimeria species in broiler facilities and could shorten the longevity of improved drug sensitivity observed in this study.

Monitoring coccidia in commercial broiler chicken flocks in Ontario: comparing oocyst cycling patterns in flocks using anticoccidial medications or live vaccination.

Coccidiosis, the parasitic disease caused by Eimeria spp., is controlled during broiler chicken production through the inclusion of in-feed anticoccidial medications. Live-coccidiosis vaccination has become an increasingly common alternative to these medications. Monitoring infections with Eimeria spp. in flocks can be accomplished through determining the concentration of oocysts excreted in the fecal material (i.e., oocysts per gram; OPG). The purpose of our study was to sample commercial Ontario broiler chicken flocks at various times of the year to determine weekly OPG counts for flocks that use either an in-feed anticoccidial medication or a live-coccidiosis vaccine. Weekly sampling of 95 flocks from placement to market permitted documentation of oocyst cycling patterns typical of conventional and antibiotic-free flocks, and variation of these patterns in summer and winter. Medicated flocks had higher and later peak oocyst shedding compared with vaccinated flocks. Flocks reared in the summer peaked in oocyst shedding earlier than flocks reared in the winter. Despite what appears to be poorer coccidiosis control in the medicated flocks, the performance data were similar for these flocks compared with vaccinated flocks. This is the first study describing typical patterns of parasite shedding in Ontarian commercial broiler chicken flocks; these data will provide a baseline of expected Eimeria spp. infections in Canadian broiler chicken flocks to ensure optimal coccidiosis prevention.

Non-infectious corneal vascularization.

A 26-year-old white female presented with a complaint of tearing and light sensitivity of several weeks duration. Examination revealed a translucent foreign body embedded in the temporal aspect of the right cornea. The fragment embedded in the cornea produced localized corneal inflammation and vascularization. The foreign body was removed and the eye was pressure patched overnight. One day after removal of the foreign body, the eye was white and quiet and the patient was comfortable. This case report represents a unique in vivo observation into the pathogenesis of non-infectious corneal vascularization.

Influence on adipocyte plasma membrane bound protein kinase by feedback regulator.

Protein kinase, phosphodiesterase and adenylate cyclase of plasma membrane of adipocytes and the effect of the feedback regulator (FR) on these three enzymes was measured and compared. The basal level ratio of adenylate cyclase to phosphodiesterase to protein kinase was 1:1.9:3.0. Epinephrine and/or FR alters this ratio. FR stimulated protein kinase activity up to 3 fold in the presence of a wide range of enzyme concentrations, 5-50 mug membrane protein/tube. The concentration of FR effective for stimulation of membrane protein kinase was much greater than that needed for inhibition of adenylate cyclase and phosphodiesterases. The inhibition by FR on adenylate cyclase was the most potent effect among the 3 enzymes. 1 U (or 2 U/ml) of FR inhibited 50% of the adenylate cyclase activity in a defined system. The maximum effective concentration of FR for stimulation of membrane protein kinase was greater than 10 U/ml. Histone type 11A was the best substrate for protein phosphorylation so far observed. The FR stimulatory effect was observed at all substrate concentrations used ranging from 1-5 mg/ml. A NaF concentration curve shows that 15 mM NaF gave maximum phosphorylation. The stimulatory effect of FR was observed both in the presence and absence of NaF. Protein kinase of adipocyte plasma membrane was mainly cAMP-independent. The effect of FR (20 U/ml) in stimulation of protein phosphorylation was much greater than that of cAMP (1 X 10(-6) M). The cAMP and FR effects seemed to be additive. Preincubation of plasma membrane with FR in the absence of ATP resulted in no decrease but slight increase in protein kinase activity. A shift in protein kinase, phosphodiesterase and adenylate cyclase ratios by FR suggests the regulatory role of FR in cAMP metabolism in adipocytes.