Performance, egg quality, and liver lipid reserves of free-range laying hens naturally infected with Ascaridia galli

Performance, egg quality, and liver lipid reserves of free-range laying hens naturally infected with Ascaridia galli N. Sharma,∗,† P. W. Hunt,†,1 B. C. Hine,† N. K. Sharma,∗ A. Chung,∗ R. A. Swick,∗ and I. Ruhnke∗ ∗

School of Environmental and Rural Science, University of New England, Armidale, NSW 2351, Australia; and † F. D. McMaster Laboratory, Chiswick, Commonwealth Scientific and Industrial Research Organization (CSIRO), Armidale, NSW 2350, Australia hens of the low infection group (23.8 ± 4.0 and 1,820 ± 450 eggs/g) (P < 0.01) and similar worm counts to PC hens (34.4 ± 4.0 and 2,918 ± 474) (P > 0.05). Egg production, egg mass, feed intake, and feed conversion ratio (FCR) were not affected by A. galli infection (P > 0.05). Egg quality parameters (egg weight, shell reflectivity, shell weight, shell thickness, shell percentage, shell breaking strength, deformation, albumen height, Haugh unit, and yolk score) were not affected by A. galli infection (P > 0.05). Highly infected hens had lower liver lipid content (2.72 ± 0.51 g) compared to uninfected hens (4.46 ± 0.58 g, P < 0.01). The results indicate that exposure to ranges contaminated with A. galli resulted in infection of the ranging hens, but this did not affect egg production or egg quality. Infection with A. galli lowered the liver lipid reserves of the host significantly, suggesting infected hens use more energy reserves for maintenance and production.

ABSTRACT A study was conducted to determine the performance, egg quality, and liver lipid reserves of laying hens exposed to ranges contaminated with Ascaridia galli. Sixteen-week-old Lohmann Brown laying hens (n = 200) were divided into 4 treatments with 5 replicates containing 10 hens per pen. Hens of treatment 1 [negative control (NC)] ranged on a decontaminated area, and hens of treatments 2 (low infection) and 3 (medium infection) ranged on areas previously contaminated by hens artificially infected with 250 and 1,000 embryonated A. galli eggs, respectively. The hens of treatment 4 [positive control (PC)] ranged on areas previously contaminated by hens artificially infected with 2,500 embryonated A. galli eggs, and in addition these hens were orally inoculated with 1,000 embryonated eggs. Results indicated that hens of the medium infection group had a higher number of intestinal A. galli worms and A. galli eggs in the coprodeum excreta (43.9 ± 4.0 and 3,437 ± 459 eggs/g) compared to

Key words: chicken, health, nematode, parasite, poultry 2018 Poultry Science 0:1–8 http://dx.doi.org/10.3382/ps/pey068

INTRODUCTION

allow infections to be transmitted between sequential flocks of hens utilizing the same range (Hoglund and Jansson, 2011; Tarbiet et al., 2015). Parasitic infections are a major threat to the Australian egg industry where the retail demand for free-range eggs is continuously increasing with a current (grocery) market value share of 55% (AECL, 2016). Equally important are increased consumer concerns regarding food quality. Thus, it is important to study the effect of A. galli infection on hen performance and egg quality when laying hens are allowed to range freely in the external environment. Previous studies have been undertaken to evaluate the effect of external and internal parasites, as well as viral infections on egg quality. An external parasite, the northern fowl mite, was found to degrade the internal quality of eggs including yolk color (Vezzoli et al., 2016). Similarly, Newcastle disease virus was found to decrease the albumen score, weight of eggs and increase mortality (Quinn et al., 1956). Other viral diseases such as Avian influenza and infectious bronchitis are also

Ascaridia galli infection is a re-emerging disease in countries where traditional caged bird facilities are being replaced with a barn or free-range system (Kaufmann et al., 2011; Thapa et al., 2015). A. galli infections have been reported to increase mortality, damage the intestinal tract, and impair digestion through limited absorption of nutrients in laying hens (Hurwitz et al., 1972; Luna-Olivares et al., 2015; Hinrichsen et al., 2016). Indirect losses include impairment of humoral immune responses after vaccination against other pathogens and increased severity of bacterial infections such as Salmonella spp. and Escherichia coli (Eigaard et al., 2006; Permin et al., 2006; Pleidrup et al., 2014). The shedded eggs of A. galli are highly resistant to external environmental conditions, which  C 2018 Poultry Science Association Inc. Received September 28, 2017. Accepted February 8, 2018. 1 Corresponding author: [email protected]

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SHARMA ET AL.

known to reduce eggshell quality (Butcher and Miles, 2003). Reid et al. (1973) reported observing mature A. galli worms in egg albumen, possibly due to high parasitic burden in the intestine resulting in migration of the parasites into the oviduct and becoming enshelled inside the eggs. A similar case was reported in Italy by Fioretti et al. (2005). These reports indicate that A. galli infection can impact egg quality but there is limited information in the literature to describe how prevalent this problem may be, and the risk factors are not well understood. It also has been reported that A. galli infection disrupts liver lipid metabolism and reduces dietary metabolizable energy (Walker and Farrel, 1976; Bansal et al., 2005), suggesting that A. galli infections may affect performance and liver lipid reserves of laying hens. This study was conducted to evaluate the impact of A. galli infection acquired from contaminated ranges on performance, egg quality, and liver lipid reserves in free-range laying hens.

Experimental Design The experiment employed a stratified randomized design with 5 replicate pens of 10 hens for each of the 4 treatments. The treatments were a 2 × 4 factorial arrangement with age (25, 30 wk) and infection groups (negative control, low, medium and high) as main factors. Hens of treatment group 1 served as a negative control (NC) and ranged on a decontaminated area (ranges were covered with bitumen one wk prior to hen access) and were treated with Levamisole (4 mL/hen) every 3 wk throughout the experiment. Hens assigned to treatment 2 (low infection) and treatment 3 (medium infection) ranged on the areas previously contaminated using hens artificially infected with 250 and 1,000 embryonated A. galli eggs, respectively (Sharma et al., 2018). Hens of treatment 4 were orally inoculated at 18 wk of age with 1,000 embryonated eggs and served as a positive control (PC). These hens ranged on an area previously contaminated by hens artificially infected with 2,500 embryonated A. galli eggs as described below.

MATERIALS AND METHODS Ethics Approval

Preparation of Infective Oral Inoculums

All experimental procedures were approved by the Animal Ethics Committee of the University of New England, Australia (approval No AEC 16–075). Hens were housed in accordance with the Model Code of Practice for the Welfare of Animals, Australia (Primary Industries Standing Committee, 2002).

To prepare embryonated A. galli eggs for the hens in treatment group 4, mature A. galli nematodes were collected from the intestines of naturally infected laying hens on a commercial farm. The mature nematodes were washed in sterile phosphate-buffered normal saline and transferred into Roswell Park Memorial Institute (RPMI) media at 37◦ C (with 0.1% 100 units/mL penicillin, 100 μg/mL of streptomycin, and 250 ng/mL amphotericin B) and cultured for 3 d, changing the media every 24 hours. Eggs shed into the media were collected by centrifugation after each 24-hour period, and concentrated eggs were re-suspended in 0.1 N H2 SO4 and kept at 26◦ C for up to 6 weeks. Embryonation was judged to have occurred after 3 wk of culture when fully formed nematodes were visible within the shell. For inoculation, the embryonated eggs were diluted in an equal volume of 0.05 M NaHCO3 and then diluted in 0.05 M NaCl to the desired concentration. Hens in treatment 4 were orally inoculated using a 16-gauge needle as described by Sharma et al. (2017).

Birds and Housing Two hundred 16-weekold Lohmann Brown laying hens were obtained from a commercial pullet rearing facility. The hens were treated with Levamisole (4 mL/hen per os) on the d of arrival and kept in the hen houses for 2 wk until acclimatized before being released onto the ranges. The hens were randomly assigned to 20 identically constructed pens, with 10 hens per pen. The pens were equipped with one gravity-filled round feeder, one bell drinker, 2 wooden nest boxes, and a wooden perch of 1 m length. The indoor area was structured with 9 m2 of slat floor and 1 m2 of solid floor covered with wood shavings. Lighting programs followed breeder recommendations (Lohmann, 2016). Hens were individually numbered using leg bands and provided with ad libitum feed (commercial top layer mash, Barastoc, Ridley, Tamworth, Australia) and water. Each pen had a main door and a pop hole leading to the range. The pop holes were opened every morning at 8 am and closed between 5 and 6 pm in the evening, allowing hens to access the ranges for minimum of 9 h every day. Each housing unit covered 10 m2 range area, in accordance with the minimum industry Australian standard of 10,000 hens/ha (CAF, 2016).

Infection Intensity Infection intensity was measured by counting eggs of A. galli in the excreta collected from each pen. Excreta samples were collected at 2, 4, and 6 wk after initial range access, followed by weekly collection until the experiment was terminated 14 wk later. Excreta samples also were collected from individual hens directly from the coprodeum at the time of necropsy (when hens were 30 wk of age), 14 wk after range access. The number of A. galli eggs was evaluated using a modified McMaster flotation method adapted from MAFF (1986). Four grams of excreta were ground

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ASCARIDIA GALLI IN FREE-RANGE LAYING HENS

and placed in a 60 mL McMaster jar with 10 mL of water. This was soaked for 30 min, and saturated NaCl solution was added to a total volume of 60 mL. The solution was stirred and the suspension loaded into McMaster egg counting chambers (0.3 mL), and eggs were counted using a microscope at 40x magnification (stereo compound microscope Olympus CX31, Tokyo, Japan). The intestinal adult A. galli worms were counted in all hens at 30 wk of age by direct observation of the intestine, split longitudinally using blunt-ended scissors.

Performance and Egg Quality Eggs from each pen were collected daily to record the number and weight of eggs until hens were sacrificed at 30 wk of age. Average feed intake per pen was measured (25 and 30 wk of age) by subtracting the total feed offered from the residual leftover feed. Individual body weight also was measured when hens were 25 and 30 wk of age. Productivity (eggs/hen/day), egg mass (egg weight∗ daily egg production), and feed conversion ratio (feed intake/egg mass) were calculated when hens were 25 and 30 wk of age. At 30 wk of age (12 wk after range access), 6 eggs were randomly chosen from each pen to analyze their quality on the same day. The external egg quality parameters included egg weight, shell weight, shell reflectivity, shell thickness, deformation, and shell breaking strength. Internal egg quality parameters included albumen height, Haugh unit, and yolk color score. Eggshell and egg internal quality were measured using TSS (Technical Services and Supplies, York, UK), shell reflectivity (TSS-QCR), egg weight and shell weight (TSSQCBi), shell breaking strength (N) by quasi-static compression and shell deformation (TSS QC-SPA-50 N load cell), shell thickness (custom-built gauge based on Mitutoyo dial comparator gauge, Model 2109–10 Kawasaki, Japan), and shell percentage calculated from shell weight and egg weight. To determine shell breaking strength, eggs were presented horizontally in TSS QCSPA equipment, and force was applied directly to the egg equator. Albumen height (mm), Haugh unit (TSS QCH), and yolk color (TSS-QCC) were also measured using standard procedures.

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A. galli worms in their intestines (NC group). Liver lipids were extracted using a Soxhlet apparatus as described by Folch et al. (1957). The liver samples were thawed, and 20 ± 0.5 g of the sub-samples were cut and freeze dried (Christ, Alpha 1–2 LD plus, Osterode am Harz, Germany) for 72 hours. Samples were then oven dried at 50◦ C for 48 h and weighed. They were then ground and wrapped in No. 1 Whatmann filter paper of size 185 mm (Sigma-Aldrich, St. Louis, MO) and packed in a Soxhlet basket. The Soxhlet unit was assembled in a fume cupboard, a 5 L solvent flask was filled with chloroform, and the basket containing the liver samples was placed in the Soxhlet chamber. After 72 h of ether extraction, samples were dried in an oven at 80◦ C for 24 hours. The total liver lipid content was then calculated by multiplying the liver lipid proportions by the liver weight.

Statistical Analysis The data were analyzed using JMP statistical software v. 8 (SAS Institute Inc, Cary, NC) to test the main effects of age, treatment, and their interaction. Performance parameters were subjected to 2 × 4 factorial arrangements. The experimental unit was the pen. For performance parameters measured over time, data were subjected to a two-way ANOVA with repeated measures, and difference between group means were assessed by Tukey’s HSD test at a probability level of 0.05. Fixed effects used were treatment, age, and their interaction. To compare the parameter difference between infected (low, medium, PC) and non-infected groups (NC), in the case of liver weight and liver lipid content, a t test was used. Egg and worm count data were not normally distributed and thus were cube root transformed for analysis. The relationship between A. galli worms counted in the intestine and the number of A. galli eggs obtained from the coprodeum were investigated by linear regression analysis using JMP software v. 8 (SAS Institute Inc, Cary, NC).

RESULTS Infection Intensity

Liver Fat Extraction At 30 wk of age, the individual body weight of all hens (n = 200) was recorded. The hens were sacrificed by electrical stunning followed by cervical dislocation. Eviscerated body weight was taken after removing viscera. Carcasses were then dissected to sample individual components. Whole liver was collected from individual hens, weighed, and stored at –20◦ C until further analysis. Liver weight relative to body weight of hens was calculated. For liver lipid determination, liver samples were selected from highly infected hens harboring more than 40 mature A. galli worms in their intestines (n = 60), and from uninfected hens (n = 60) with no mature

The effect of different levels of A. galli infection on excreta egg counts of laying hens at 25 and 30 wk of age (collected from pens) are presented in Table 1. The number of A. galli eggs in the excreta (egg/g of excreta) was significantly higher (P < 0.001) in hens of the medium infection group compared to hens of the low infection group but similar to hens of the PC. No A. galli eggs were detected in the excreta of hens in the NC group at any time. The effect of A. galli infection on individual hen intestinal A. galli worm counts and A. galli eggs obtained from the coprodeum content are presented in Table 2. The hens of the medium infection group had higher

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2.2 0.68 < 0.01 0.95 1.03 0.15 < 0.001 0.72 P-value

SEM Trt Age Trt × Age

4 0.20 < 0.001 0.46

1.74 0.43 < 0.001 0.84

2.252 2.304 60.4 61 58.6 59.7 58.1 59.0 138 140 119 127

52.9 55.1

2.103 60.4 60.0 57.6 55.1 137 113

0.074 0.26 < 0.01 0.28

90.3 92.3 2.381 2.383

92.1 2.382

25 90.1 30 2.373 25 2.311 30 60.2 25 58.5 30 56.6 25 52.8 30 134 25 121

Negative Treatments control (Trt) Positive control Low infection Medium infection

Weeks of age

# Where a significant overall treatment group effect was observed (independent of age when hens were tested), treatment group means that differed significantly are depicted using different superscripts (P < 0.05). SEM is average of 25 and 30 weeks. All measurements were done at pen level (n) = 5 replicates per pen. 1 Egg mass = egg weight∗percentage egg production. 2 FCR = Feed conversion ratio, feed intake/egg mass.

16 < 0.01 < 0.001 0.86

1.6 < 0.001 0.86 0.71

302.1b 1228.5a 167.3 1481.3 1921a 1840c 96.0 97.0

1884 1816

1958 1863

493.0 1000

746.1a,b 773.6 1864b,c 95.4

1838

1920

531.4

Mean# 0.0c 30 0.0 25 0.0 Mean# 1886a,b 30 1889 30 94.0

25 1852

Body weight (g) Productivity (%) FCR2 Egg weight (g) Egg mass1 (g) Feed intake (g/hen/d) Parameters

Table 1. Main effect of different levels of A. galli infection on performance and excreta egg counts of free-range laying hens at 25 and 30 wk of age.

Excreta egg counts (eggs/g)

SHARMA ET AL.

(P < 0.001) A. galli worm counts in the intestine (43.9 ± 4.0 worms/hen) compared to hens of the low infection group (23.8 ± 4.0 worms/hen) but similar worm counts to those of PC hens (34.4 ± 4.0 worms/hen). The hens in the NC group had no worms in their intestines. Similarly, the hens of the medium infection group had higher (P < 0.001) egg counts in the coprodeum (3437 ± 459) compared to those in the low infection group (1820 ± 449), but similar egg counts to hens of the PC group (2914 ± 474). No A. galli eggs were observed in the excreta obtained from hens of the NC group. At 30 wk of age, the intestinal worm counts and coprodeum excreta A. galli egg counts were highly correlated (R2 = 0.80) and had a significant positive linear relationship (P < 0.01) (Figure 1).

Effect on Performance, Eviscerated Weight, and Liver Lipid Reserve The effect of A. galli infection on feed intake, egg mass, egg weight, FCR, productivity, and body weight are presented in Table 1. There were no effects of treatments on feed intake, egg mass, egg weight, FCR, productivity, or body weight at 25 and 30 wk when assessed at the pen level (P > 0.05). All hens started laying eggs at 19 wk of age and reached the peak of production at 26 wk of age with eggs produced by 95% of hens daily, and then the level of production remained similar until 30 wk of age when the experiment ended (Figure 2). There were no differences in body weight, eviscerated body weight, liver weight, or relative liver weight among the hens of all treatment groups at 25 and 30 wk of age (Table 2). Relative liver weight was 2.23 ± 0.06 g/kg in hens of the NC group, whereas it was 2.07 ± 0.06 g/kg, 2.09 ± 0.06 g/kg and 2.06 ± 0.06 g/kg in hens of the PC, low, and medium infection groups, respectively. When all infected groups (pooled) were compared to the uninfected NC group (Figure 3), relative liver weight in hens of the NC group and hens of the infected group did not differ (2.23 ± 0.21 g/kg, vs. 2.06 ± 0.60 g/kg, respectively, P = 0.21). In a previous study, we observed a range of liver lipid contents in hens, and there was a suggestion that the lipid content was lower in hens with a high level of infection (Sharma et al., 2018). Therefore, in this study we decided to compare a group of hens with high worm counts, greater than or equal to 40 worms, with hens from the NC group. Analysis demonstrated that the liver lipid content was higher in the hens from the NC group (4.46 ± 0.22 g) compared to those from the selected hens with high infections (2.72 ± 0.65 g) (P < 0.001).

Effect on Egg Quality The effect of various levels of A. galli infection in laying hens on external egg quality parameters (egg weight, shell weight, shell reflectivity, shell thickness,

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ASCARIDIA GALLI IN FREE-RANGE LAYING HENS

Table 2. Effect of A. galli infection on body weight, eviscerated weight, liver weight, intestinal worm counts, and coprodeum egg counts of free-range laying hens at 30 wk of age.

Parameters Treatment

Body weight (g)

Eviscerated body weight (g)

1899 1848 1911 1868

1558 1513 1564 1530

Negative control Positive control Low infection Medium infection

P-value

Eviscerated yield (%)

Liver weight (g)

82.1 81.9 82.0 81.9

42.4 38.3 39.7 38.4

Relative liver weight (g/kg)

Intestinal worm counts

Coprodeum egg counts

2.23 2.07 2.09 2.06

0.00c 34.4a,b 23.8b 43.9a

0.00c 2914.8a,b 1820.0b 3437.2a 459.09

SEM

17.87

17.08

0.69

1.35

0.06

3.96

Treatment

0.093

0.162

0.990

0.151

0.213

< 0.001

< 0.001

Means in each row for each factor with different superscripts differ significantly (P < 0.05). Total number of hens for all parameters measured were (n) = 200, coprodeum egg count (n) = 80 a–c

Table 3. Effect of different levels of A. galli infection on egg weight and egg quality of free-range laying hens at 30 wk of age.

Parameters Treatment

Negative control Positive control Low infection Medium infection SEM

P-value

Treatment

Egg weight (g)

Shell weight (g)

Shell reflectivity (%)

Shell thickness (mm)

Shell percentage

Breaking shell strength (N)

59.7 60.8 60.1 61.5 0.56

6.07 6.24 6.29 6.35 0.07

19.1 19.5 19.8 18.8 0.50

0.44 0.43 0.44 0.44 0.004

10.2 10.3 10.5 10.3 0.15

45.1 45.5 45.7 46.0 1.09

0.154

0.082

0.540

0.851

0.433

0.932

Deformation 0.28 0.28 0.30 0.28 0.01 0.241

Albumen height (mm)

Haugh unit

Yolk score

10.1 10.7 10.3 10.6 0.17

99.9 102.2 100.5 102.0 0.75

10.1 10.1 10.0 10.3 0.19

0.064

0.111

0.632

Parameters were measured in total number of eggs (n) = 120.

Intestinal worm count (n)

80 y = 0.0104x + 4.0178

70 60

R² = 0.8031, P < 0.01

50 40 30 20 10

0 0

1000

2000 3000 4000 5000 Coprodeum egg count (eggs/g)

6000

Figure 1. Correlation between A. galli worm in intestine and A. galli eggs in coprodeum content of laying hens at 30 wk of age.

shell percentage, shell breaking strength, and deformation) and internal egg quality parameters (albumen height, Haugh unit, and yolk score) are presented in Table 4. A. galli infection had no significant effect on the external or internal egg quality parameters measured (P > 0.05). No worms were observed during the processing of eggs for internal egg quality or at any other time. The average egg weight was 60 g ± 0.56 at 30 wk of age. The average shell thickness, shell reflectivity, shell breaking strength, height of albumen, and yolk score for eggs in all the groups were 0.4 ± 0.004 mm, 19.0 ± 0.50%, 46.0 ± 1.09 N, 10 ± 0.17 mm, and 10 ± 0.19, respectively.

DISCUSSION

havior and may increase animal welfare, access to litter and outdoor areas also poses an increased risk of endoparasite infections (Hinrichsen et al., 2016). Evidence suggests that hens become infected if placed in an environment where residual infective A. galli eggs are present (Hoglund and Jansson, 2011). However, it is not clear whether the transmission on a commercial farm would result in similar infection intensity as observed in this study. This study was conducted 17 wk after the ranges were contaminated with A. galli shedding hens, which ranged there for 20 weeks. This design allowed us to mimic the commercial egg production while being able to monitor individual hens with a required amount of replicates. The mean intestinal worm counts and excreta A. galli egg counts in hens of the infected group were significantly higher in the current study compared to the results obtained in a previous artificial infection study (Sharma et al., 2018). This suggests that the infection intensity acquired through natural infection may be higher than that achieved through experimental (artificial) infection. The eggs of A. galli can tolerate external environmental conditions, e.g., A. galli eggs could survive at freeze-thaw cycle (30 min to 12 h), and 4% of A. galli eggs developed into infective eggs at 35◦ C temperature (Tarbiat et al., 2015). Birds are the main host of the direct life cycle and can get infected after they ingest embryonated A. galli eggs. Prevalence studies on commercial organic and free-range laying hens have shown A. galli infection prevalence of up to 98% and 10 times higher infection intensity (intestinal A. galli counts and excreta egg counts) compared to hens that were artificially infected (Gauly et al., 2005;

In free-range egg production systems, laying hens are allowed to range in an open environment. While this production system promotes normal physiological beDownloaded from https://academic.oup.com/ps/advance-article-abstract/doi/10.3382/ps/pey068/4941979 by University of Glasgow user on 30 April 2018

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SHARMA ET AL.

Relative liver weight and liver fat (g)

Figure 2. Effect of A. galli infection on egg production from 19 to 30 wk of age. a

5 4 3

b a

a

2 1 0 Relative liver weight (g/kg) Negative control

Liver fat (g) Infected

Figure 3. Relative liver weight (liver weight/body weight) and total liver fat content of A. galli-infected and non-infected laying hens at 30 wk of age. Liver weight relative to body weight was measured in all hens (n = 200), and liver lipid was measured in negative control vs. infected hens (harboring more than 40 mature A. galli worms in their intestine) (n = 120).

Kaufmann et al., 2011; Da¸s et al., 2010; Thapa et al., 2015). It is expected that the daily natural foraging behavior continuously exposes hens to A. galli eggs and that these gradually accumulating inoculations can establish higher infection intensities compared to delivering embryonated eggs in a few bolus doses. Further, hens in this study were observed on the ranges daily. This may have resulted in a continuous daily uptake of embryonated A. galli eggs and subsequently explain the higher infection compared to artificial infection. The results obtained from this study demonstrate the potential transmission of A. galli infection from one flock of hens to the next even after a 17-week rest period between batches of hens in which daily minimum temperatures frequently fell below freezing. This emphasizes the need for research to develop effective strategies for reducing A. galli contamination on ranges. In the current study, hens of the medium infection group had higher intestinal worm counts and coprodeum egg counts compared to hens of the low infection group but similar to hens of the PC group, suggesting that the level of contamination of the range is proportional to

the infection level acquired in hens introduced to contaminated ranges. The current study revealed no differences in feed intake, body weight, or FCR measured at 25 and 30 wk of age among treatment groups. In contrast, a number of studies have reported increased feed intake, reduced body weight, and increased mortality in A. galli-infected laying hens (Ikeme, 1971; Gauly et al., 2007; Hinrichsen et al., 2016). This discrepancy might be explained by the fact that hens evaluated by the previous authors were older (37 wk of age). Increased feed intake was suspected to compensate for maintaining body weight and egg production (Gauly et al., 2007). In agreement with our findings, Das et al. (2012) reported no effect of A. galli infection on feed intake of growing layers; however, the researchers reported that infection did result in reduced body weight gain. Further, in the current study, no clinical signs or mortalities were observed throughout the experimental period, which is similar to the findings of Dahl et al. (2002). The experimental period investigated here may have been too short to allow severe clinical signs to develop and cause mortalities in birds. It has been reported that dietary metabolizable energy is reduced by 4% in A. galliinfected broilers (Walker and Farrell, 1976). Therefore, in the current study, infected hens would have needed to increase feed intake or reduce their body weight to meet their additional energy needs; however, this was not the case. Interestingly, the lower liver lipid content in A. galli-infected hens compared to the hens in the NC group in the current study may indicate that the hens utilized the energy reserves stored in the liver to maintain performance in the face of infection. Further research in a full laying cycle is recommended to see the impact of liver lipid reserve depletion on hen health and performance. Hens from all treatment groups started laying at 19 wk of age, suggesting that A. galli infection had no impact on the onset of lay regardless of the infection level. Even if hens would have taken up embryonated eggs from the range on their first d of

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range access (when hens were 18 wk of age), the embryonated eggs would have just hatched and started their mucosal phase. This phase is usually accompanied by minor impact on the immune system. It therefore may have been too early to see a clinical impact on the host, such as a delayed onset of lay. The egg production and egg weight were not affected by A. galli infection up to 30 wk of age. In support of our findings, Dahl et al. (2002) observed no effect of A. galli infection on egg production to 31 wk of age when hens were infected at 18 wk of age. Previous studies have reported a decline in egg production in layers due to A. galli infection (Permin et al., 1998); however, the result observed in that study might be related to the relatively high age of chickens when infected (Kerr, 1955). In commercial settings when pullets are raised on the floor, infection due to used litter or external contamination may occur at any age, and infection may be established at 16 wk of age when transition to the layer house occurs. Hens obtained for the current research were raised in an aviary system with fresh litter exposure, no access to the outdoors, and regular anthelmintic treatment. In addition, all hens were treated with Levamisole when enrolled in the study. It is possible that A. galli infection during the early laying period, as we observed, might not affect performance until the mid to late stages of the laying cycle, well past the 14 wk over which we conducted our study. This requires further investigation using experiments with a longer duration. Various factors such as hen breed, strain within the breed, age, nutrition, stress, disease, and production systems are thought to affect egg quality in laying hens (Roberts, 2004). Maintaining egg quality is of utmost importance, especially when hens are raised in a freerange production system where chances of parasitic infections are higher than in a conventional cage system. In a recent study, it was reported that external egg quality variables such as egg weight, shell weight, and shell thickness were higher in hens raised in conventional cages as compared to hens raised in a free-range system (Samiullah et al., 2014). For example, the yolk color was denser in caged eggs compared to free-range eggs from hens that were provided identical diets. Total bacterial load was higher in free-range eggs compared to caged eggs (Samiullah et al., 2014). To our knowledge, the effect of parasitic infection on egg quality in laying hens is largely unknown. In the current study, egg quality parameters (both external and internal) were measured. Results indicated that there was no effect of A. galli infection on any of the egg quality parameters in hens to 30 wk of age. Further investigations are required to monitor egg quality of A. galli-infected hens throughout the production cycle. In conclusion, A. galli infection acquired from contaminated ranges had no effect on performance or egg quality from the point of lay until 30 wk of age. However, lowered liver lipid reserves in infected hens indicated that these hens may have used the energy reserves

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for maintenance and production. Further studies are warranted to investigate the effect of A. galli infection during the entire egg production cycle.

ACKNOWLEDGMENTS This research was conducted within the Poultry CRC, established and supported under the Australian Government’s Cooperative Research Centers Program and is a part of Poultry CRC sub-project 1.5.9. The authors would like to thank students and staff of Animal House at University of New England, Australia, for their helping hands during this project.

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