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.

The effect of alternative feeding strategies on the feeding motivation of broiler breeder pullets.

Due to genetic selection for fast growth and high breast meat yield, commercial strains of broiler chickens and broiler breeders are predisposed to high feed intake; however, feeding broiler breeders ad libitum impairs their health and reproductive performance. Broiler breeders are feed-restricted throughout rearing to maintain health and performance, yet feed restriction results in hunger, feeding frustration and lack of satiety. The objective of this study was to examine the effect of alternative feeding strategies, including feed additives (separately or combined) and a fixed non-daily feeding schedule, on the feeding motivation and welfare of broiler breeders during rearing. At 3 weeks of age, 180 Ross 308 breeder pullets were allocated to 90 cages and fed with one of five isocaloric treatments: (1) daily control diet (control), (2) daily calcium propionate diet (CaP), (3) daily soybean hull diet (SBH), (4) daily alternative diet (alternative: CaP + SBH) and (5) 4/3 control diet (four on-feed days and three non-consecutive off-feed days per week). The CaP diet included calcium propionate at 1.4% from 3 to 6 weeks of age, and at 3.2% from 7 to 12 weeks of age, and the SBH diet contained soybean hulls included at 40%. The alternative diet included both soybean hulls and calcium propionate at the same inclusion rate as the SBH and CaP diets, respectively. Pullets were weighed and scored for feather coverage every week. A feed intake test was conducted at 3, 4, 8, 10 and 11 weeks of age for 10 min during on- and off-feed days. At 12 weeks of age, feather samples were analysed for fault bars. Data were analysed using linear mixed regression models, with cage nested in the models and age as a repeated measure. At 4 weeks of age, pullets fed soybean hull-enriched diets (SBH and alternative diets) and those on the 4/3 schedule had lower feed intake than control pullets (P = 0.02). Feathers from pullets fed the SBH diet had fewer fault bars than those fed the CaP diet (P = 0.04). The results indicated that the inclusion of soybean hulls (alone or combined with calcium propionate) and a 4/3 feeding schedule can reduce feeding motivation of broiler breeders during early rearing.

The effect of alternative feeding strategies for broiler breeder pullets: 2. Welfare and performance during lay.

Feeding broiler breeders to satiety has negative consequences on their health and reproduction. Alternative feeding strategies during rearing can improve welfare, although their implications during lay are not well understood. The objective was to examine the effect of rearing feeding treatments on the reproductive performance and feeding behavior of broiler breeders under simulated commercial conditions. At 3 wk of age, 1,680 Ross 308 pullets were allocated to 24 pens under 1 of 4 isocaloric treatments: 1) daily control diet; 2) daily alternative diet (40% soybean hulls and 1 to 5% calcium propionate); 3) 4/3 control diet (4 on-feed days, 3 non-consecutive off-feed days per week); and 4) graduated control diet. Feeding frequency of the graduated treatment varied with age and finished on a daily basis. At 23 wk of age, group sizes were adjusted to 40 hens, and 5 mature Yield Plus Males roosters were introduced to each pen. Pens were under the same daily feeding management and same diet during lay. The performance of broiler breeders (growth rate, body weight uniformity, and reproductive performance) was determined until 64 wk of age. At the end of lay, feeding motivation was examined with a feed intake test and a compensatory feeding test. Data were analyzed using linear mixed regression models, with pen nested in the models and age as a repeated measure. The laying rate of hens reared on the graduated treatment decreased slower compared to control hens, resulting in a higher cumulative egg production (178.2 ± 3.8 eggs/hen) than control hens (165.2 ± 3.8 eggs/hen, P < 0.01) by 64 wk of age. Hens reared on non-daily feeding treatments laid lighter eggs with relatively heavier yolks and had higher feed intake at the end of lay than hens fed daily during rearing (P = 0.02). In conclusion, rearing feeding treatments impacted the growth rate and body weight uniformity during lay, feeding motivation at the end of lay, and the laying rate and hatchability depending on hens' age.

Opportunities for exercise during pullet rearing, Part II: Long-term effects on bone characteristics of adult laying hens at the end-of-lay.

Osteoporosis in laying hens has been a production and welfare concern for several decades. The objective of this study was to determine whether differing opportunities for exercise during pullet rearing influences long-term bone quality characteristics in end-of-lay hens. A secondary objective was to assess whether differing opportunities for exercise in adult housing systems alters bone quality characteristics in end-of-lay hens. Four flock replicates of 588 Lohmann Selected Leghorn-Lite pullets were reared in either conventional cages (Conv) or an aviary rearing system (Avi) and placed into conventional cages (CC), 30-bird furnished cages (FC-S), or 60-bird furnished cages (FC-L) for adult housing. Wing and leg bones were collected at the end-of-lay to quantify bone composition and strength using quantitative computed tomography and bone breaking strength (BBS). At the end-of-lay, Avi hens had greater total and cortical cross-sectional area (P < 0.05) for the radius and tibia, greater total bone mineral content of the radius (P < 0.001), and greater tibial cortical bone mineral content (P = 0.029) than the Conv hens; however, total bone mineral density of the radius (P < 0.001) and cortical bone mineral density of the radius and tibia (P < 0.001) were greater in the Conv hens. Hens in the FC-L had greater total bone mineral density for the radius and tibia (P < 0.05) and greater trabecular bone mineral density for the radius (P = 0.027), compared to hens in the FC-S and CC. Total bone mineral content of the tibia (P = 0.030) and cortical bone mineral content of the radius (P = 0.030) and tibia (P = 0.013) were greater in the FC-L compared to the CC. The humerus of Conv hens had greater BBS than the Avi hens (P < 0.001), and the tibiae of FC-L and FC-S hens had greater BBS than CC hens (P = 0.006). Increased opportunities for exercise offered by the aviary rearing system provided improved bone quality characteristics lasting through to the end-of-lay.

Opportunities for exercise during pullet rearing, Part I: Effect on the musculoskeletal characteristics of pullets.

Increased load-bearing exercise improves bone quality characteristics in a variety of species, including laying hens. Providing increased opportunities for exercise during the pullet rearing phase, a period of substantial musculoskeletal growth, offers a proactive approach to reducing osteoporosis by improving bone composition. The main objective of this study was to determine whether differing opportunities for exercise during rearing influences pullet musculoskeletal characteristics. Two flock replicates of 588 Lohmann Selected Leghorn-Lite pullets were reared in either standard, conventional cages (Conv) or an aviary rearing system (Avi) from day-old chicks until 16 wk of age. The keel bone and the muscles and long bones of the wings and legs were collected at 16 wk to measure muscle growth differences between rearing treatments and quantify bone quality characteristics using quantitative computed tomography (QCT) and bone breaking strength (BBS) assessment. Keel bone characteristics and muscle weights were adjusted for BW and analyses for QCT and BBS included BW as a covariate. At 16 wk of age, rearing system had an effect on the majority of keel bone characteristics (P < 0.05). The length of the keel metasternum, caudal tip cartilage length, and the overall percentage of cartilage present on the keel at 16 wk was greater in the Avi pullets compared to the Conv pullets (P < 0.01). Wing and breast muscle weights of the Avi pullets were greater than the Conv pullets (P < 0.001), but leg muscle weights were greater in the Conv pullets (P = 0.026). Avi pullets had greater total bone density, total cross-sectional area, cortical cross-sectional area, total bone mineral content, and cortical bone mineral content than Conv pullets for the radius, humerus, and tibia (P < 0.001). Avi pullets had greater BBS compared to the Conv pullets for the radius, humerus, and tibia (P < 0.01). Increased opportunities for exercise offered by the aviary rearing system increased muscle and bone growth characteristics in pullets at 16 wk of age.

Rearing system affects prevalence of keel-bone damage in laying hens: a longitudinal study of four consecutive flocks.

High flock-level prevalence of keel-bone fractures and deviations in laying hens are commonly reported across various housing systems; however, few longitudinal studies exist, especially for furnished and conventional cage systems. Load-bearing exercise improves bone strength and mineral composition in laying hens and has the potential to reduce keel-bone damage, especially if exercise is allowed during critical periods of bone growth throughout the pullet rearing phase. The objective of this study was to determine the prevalence of keel-bone damage in laying hens housed in furnished and conventional cages, and assess whether opportunities for exercise during the pullet rearing phase influenced the prevalence of keel-bone damage throughout the laying period. Four flock replicates of 588 Lohmann Selected Leghorn-Lite pullets/flock were reared in either conventional cages (Conv) or an aviary rearing system (Avi) and placed into conventional cages (CC), 30-bird furnished cages (FC-S) or 60-bird furnished cages (FC-L) for adult housing. Keel-bone status was determined by palpation at 30, 50, and 70 wk of age. Age (P < 0.001) and rearing system (P < 0.001) had an effect on the presence of keel-bone fractures. The presence of fractures increased with age, and hens raised in the Avi system had a lower percentage of fractures (41.6% ± 2.8 SE) compared to hens reared in the Conv system (60.3% ± 2.9 SE). Adult housing system did not have an effect on the percentage of keel fractures (P = 0.223). Age had an effect on the presence of deviations (P < 0.001), with deviations increasing with age. Rearing system (P = 0.218) and adult housing system (P = 0.539) did not affect the presence of deviations. Keel fractures and deviations were strongly associated with each other at all ages: 30 wk: (P < 0.001); 50 wk: (P < 0.001); and 70 wk: (P < 0.001). Increased opportunities for exercise provided by an aviary rearing system reduced the prevalence of keel-bone fractures through the end-of-lay.

Spatial Clustering of Escherichia coli with Reduced Susceptibility to Cefotaxime and Ciprofloxacin among Dairy Cattle Farms Relative to European Starling Night Roosts.

European starlings (Sturnus vulgaris) have been implicated in the dispersal of zoonotic enteric pathogens. However, their role in disseminating antimicrobial-resistant organisms through their home range has not been clearly established. The aim of this study was to determine whether starling night roosts served as foci for spreading organisms with reduced susceptibility to antimicrobials among dairy cattle farms. Bovine faecal pats were collected from 150 dairy farms in Ohio. Each farm was visited twice (in summer and fall) between 2007 and 2009. A total of 1490 samples (10 samples/farm over two visits) were tested for Escherichia coli with reduced susceptibility to cefotaxime and ciprofloxacin. Using a spatial scan statistic, focal scans were conducted to determine whether clusters of farms with a high prevalence of organisms with reduced susceptibility to cefotaxime and ciprofloxacin surrounded starling night roosts. Faecal pats 13.42% and 13.56% of samples carried Escherichia coli with reduced susceptibility to cefotaxime and ciprofloxacin, respectively. Statistically significant (P < 0.05) spatial clusters of faecal pats with high prevalence of Escherichia coli showing reduced susceptibility to cefotaxime and ciprofloxacin were identified around these night roosts. This finding suggests that the risk of carriage of organisms with reduced susceptibility to antimicrobials in cattle closer to starling night roosts was higher compared to cattle located on farms further from these sites. Starlings might have an important role in spreading antimicrobial-resistant E. coli to livestock environments, thus posing a threat to animal and public health.

A longitudinal study of feed contamination by European starling excreta in Ohio dairy farms (2007-2008).

The objectives of this study were to understand the temporal pattern of contamination of cattle feed by starling excrement on dairy farms and to evaluate the temporal pattern in recovering Escherichia coli O157:H7 or Salmonella in relation to the absolute mass of excrement recovered. A longitudinal study was conducted on 15 dairy farms in Ohio from July 2007 to October 2008. One open-topped tray filled with bird feed was placed near a cattle feeding site; bird excrement from the tray was weighed monthly for 12 consecutive months. Linear regression models with a random intercept for farm were computed to examine the association between the absolute weight of excrement recovered each month or the farm-specific standard score for weight of excrement, and month or season. Exact logistic regression was used to determine whether an association between recovering E. coli O157:H7 or Salmonella was present and the amount of excrement recovered and season. A spatial scan statistic was used to test for evidence of space-time clustering of excrement, based on the standard score for the weight of the excrement, among our study farms. A total of 5 of 179 excrement samples (2.79%) were positive for E. coli O157:H7 and 2 (1.12%) were positive for Salmonella. A significantly higher level of contamination with excrement was observed during the winter. The odds of recovering a pathogen increased with the amount of excrement recovered and decreased if the excrement was collected in the winter. A spatio-temporal cluster of contamination with excrement was detected. These findings provide basic information for future quantitative microbial risk assessments concerning the role of starlings in spreading enteric pathogens on dairy farms.

Keel fracture assessment of laying hens by palpation: inter-observer reliability and accuracy.

The objective of this study was to assess the interobserver reliability (agreement) and accuracy of keel palpation for the purpose of detecting old fractures in an end-of-lay flock of commercial laying hens. The low level of invasiveness and the relative speed at which this evaluation can be carried out lends itself well to use in a welfare audit, but only if the results are reliable and accurate from various assessors. The palpation technique first described by Wilkins and others (2004) was used to manually palpate for keel fractures. The technique was modified in that only keel fractures were considered. Eight assessors with varying laying hen experience palpated 100 live ISA Brown hens that had been in lay for 49 weeks. The hens were then euthanased and examined by dissection to establish whether there had been a keel fracture present (yes/no). The accuracy for individual assessors ranged from 87.1 to 96.8 per cent, with a mean of 91.8 per cent among all eight assessors. The interobserver reliability among all eight assessors was moderate (κ=0.44). Accuracy and κ values were 84.8 per cent and 0.41 for the first 50 hens, and 99.5 per cent and 0.47 for the last 50 hens, respectively, indicating that there was increased accuracy and agreement as the assessors became more experienced at palpation. This level of agreement, and the high level of accuracy, would make this technique an acceptable measure of keel fracture prevalence in a welfare audit.

On-farm study of human contact networks to document potential pathways for avian influenza transmission between commercial poultry farms in Ontario, Canada.

Human movements associated with poultry farming create contact networks that might facilitate transmission of avian influenza (AI) between farms during outbreaks. In Canada, no information is available about how these networks connect poultry farms. The purpose of this study was to document human contacts between commercial poultry farms in Ontario, Canada, to learn how AI might be transmitted during outbreaks. We used face-to-face interviews with people entering the farm biosecurity perimeter on four layer, one turkey and three broiler breeder poultry farms in Ontario to collect information on between-farm contacts and biosecurity practices. Over a four-day study period on each farm, a median of 10.5 people entered the farm biosecurity perimeter (range 2-31). Ninety-six per cent (111/118) of people consented to be interviewed. Of these, fifty-three per cent (59/111) had contact with one or more (median 2, degree range 1-14) other poultry farms within 72 h. A median of 25 (range 7-65) human contacts linked study farms to other poultry farms. The mean distance of between-farm contacts was 53 km. Eighty-six per cent of people who answered the biosecurity questions (94/109) reported using one or more biosecurity practices. However, on 7/8 farms, at least one person reported that they did not use any biosecurity practices. Fifty per cent of social visitors used biosecurity, whereas 96% of all other people used biosecurity. Ninety-two per cent of people that entered the poultry barns (46/50) used one or more biosecurity practices, whereas 81% of people (48/59) that did not enter the poultry barns used one or more biosecurity practices. Because our study documented farm visitors who did not use any biosecurity practices and moved between commercial poultry farms, we suggest that rapid trace-out of human movements is as important as containment zoning to limiting disease spread during an outbreak of highly pathogenic AI in Ontario.

The change in prevalence of Campylobacter on chicken carcasses during processing: a systematic review.

A systematic review was conducted to evaluate the change in prevalence of Campylobacter on chicken carcasses during processing. A structured literature search of 8 electronic databases using the key words for "Campylobacter," "chicken," and "processing" identified 1,734 unique citations. Abstracts were screened for relevance by 2 independent reviewers. Thirty-two studies described prevalence at more than one stage during processing and were included in this review. Of the studies that described the prevalence of Campylobacter on carcasses before and after specific stages of processing, the chilling stage had the greatest number of studies (9), followed by washing (6), defeathering (4), scalding (2), and evisceration (1). Studies that sampled before and after scalding or chilling, or both, showed that the prevalence of Campylobacter generally decreased immediately after the stage (scalding: 20.0 to 40.0% decrease; chilling: 100.0% decrease to 26.6% increase). The prevalence of Campylobacter increased after defeathering (10.0 to 72.0%) and evisceration (15.0%). The prevalence after washing was inconsistent among studies (23.0% decrease to 13.3% increase). Eleven studies reported the concentration of Campylobacter, as well as, or instead of, the prevalence. Studies that sampled before and after specific stages of processing showed that the concentration of Campylobacter decreased after scalding (minimum decrease of 1.3 cfu/g, maximum decrease of 2.9 cfu/mL), evisceration (0.3 cfu/g), washing (minimum 0.3 cfu/mL, maximum 1.1 cfu/mL), and chilling (minimum 0.2 cfu/g, maximum 1.7 cfu/carcass) and increased after defeathering (minimum 0.4 cfu/g, maximum 2.9 cfu/mL). Available evidence is sparse and suggests more data are needed to understand the magnitude and mechanism by which the prevalence and concentration of Campylobacter changes during processing. This understanding should help researchers and program developers identify the most likely points in processing to implement effective control efforts. For example, if contamination will occur during defeathering and likely during evisceration, critical control points postevisceration are likely to have a greater effect on the end product going to the consumer.

Temperature-related risk factors associated with the colonization of broiler-chicken flocks with Campylobacter spp. in Iceland, 2001-2004.

Our objective was to identify temperature-related risk factors associated with the colonization of broiler-chicken flocks with Campylobacter spp. in Iceland, with an underlying assumption that at minimum ambient temperatures, flies (Musca domestica) play a role in the epidemiology and seasonality of Campylobacter. At slaughter, pooled caecal samples were obtained from 792 flocks that hatched between March 15 and September 15 each year from 2001 to 2004, and cultured for Campylobacter. Daily temperature data (average, maximum and minimum temperatures) were obtained from the Icelandic Meteorological Office. We determined the closest weather station to each farm using ArcGIS 9. We then used logistic-regression models (with a random-effects term for farm) to examine associations between flock positivity and temperature-related predictors. The overall prevalence of Campylobacter was 27.4%, and the month with the highest prevalence (54.4%) was August. The final model included cumulative degree-days (CDD) above an average temperature of 4.4 degrees C and the presence of 1 or more days below a maximum temperature of 8.9 degrees C (threshold below which fly activity was expected to be substantially reduced and egg laying was not expected to occur) during the period 2-4 weeks (i.e. days 8-28) before slaughter. Below 79 CDD, the risk of flock Campylobacter colonization was generally low. Between 79 and 139 CDD, the risk increased gradually, but was lower for flocks raised during periods with 1 or more days <8.9 degrees C than flocks raised without days <8.9 degrees C. The risk increased sharply under conditions of high CDD (>139) and when the maximum temperature remained >8.9 degrees C during the period 2-4 weeks before slaughter.