Ascarid infections in laying hens kept in different housing systems

Abstract

The present study was undertaken to investigate the prevalence of ascarid infections in Swedish commercial laying hens in 2004 and 2008 following a recent nationwide change to alternative housing systems but before anthelmintics became available. Also, the influence on prevalence of farm and flock characteristics and management was studied in 2004. The results showed that the overall prevalence was significantly higher in 2008 (38%; n = 64/169) compared with 2004 (24%; n = 44/186) (P = 0.001). Ascarid infections were rare in caged flocks, including furnished (enriched) cages, both years (2.4 to 4.3%), and were significantly more common in non-cage systems in both years (16.7 to 48.6% in 2004, and 28.6 to 77.1% in 2008 depending on the housing system). There was no significant difference in prevalence between hens kept on litter indoors and free-range/organic hens. The absence of a hygiene barrier at the entrance of the house or unit increased the risk of infection (P < 0.001), which suggests that parasite eggs were introduced horizontally to the farms. The risk of infection also increased with the age of equipment used in the barn; for example, the risk increased with an odds ratio of 7.5 (95% confidence interval = 2.3 to 25) when comparing equipment 1 year old with equipment ≥7 years old. The results of this study show that ascarid infections may re-emerge following a change to alternative housing. With the impending ban on conventional battery cages in the member states of the European Union, ascarid infections are likely to increase in importance and efficient control options such as hygiene barriers should be implemented on all farms.

Introduction

Ascarid infections commonly occur in non-caged chickens around the world (Permin & Hansen, 1998). The two main ascarids of poultry, Ascaridia galli and Heterakis gallinarum, both have direct lifecycles including a faecal-oral route of transmission of their eggs, which display a high degree of environmental resilience. Adults reside in the small intestine (A. galli) or the caeca (H. gallinarum). Particularly in laying hens, A. galli may cause loss of body weight, increased feed consumption, intestinal haemorrhage, increased mortality by small intestinal obstruction and by synergistic effects with concomitant bacterial infections, and behavioural changes (Ackert, 1931; Ikeme, 1971a, b; Dahl et al., 2002; Kilpinen et al., 2005; Permin et al., 2006; Gauly et al., 2007). In contrast, H. gallinarum hardly ever causes clinical signs, but is an important transport host of the aetiological agent of blackhead, Histomonas meleagridis (McDougald, 2005).

For many years, control of ascarid infections in commercial laying hens may, at least to some degree, have been achieved by separating birds from their faeces; that is, by keeping hens in cages. Today, a large majority of commercial laying hens worldwide are still housed in conventional battery cages, despite the fact that this kind of housing is considered to fail to cater for basic welfare needs (Baxter, 1994; Craig & Swanson, 1994). In the near future all member states of the European Union face major animal husbandry changes due to implementation of the laying hen welfare regulations laid down in Council Directive 99/74/EC, which includes a ban on battery cages from January 2012 (European Commission, 1999). In Sweden, a ban on conventional battery cages was already imposed in 1999, but they were not replaced until the end of a phase-out period in late 2004. Today, all Swedish laying hens are housed in furnished (enriched) cages or in litter-based housing systems (Tauson, 2005). Hens in furnished cages are offered a minimum available space of 600 cm² per bird, plus a shared 200 cm² nest and 200 cm² dust bathing area, and perches. Furnished cages in Sweden normally house eight to 10 hens, and if kept in traditional single-tiered floor production, hens are provided with a litter area, perches and nests. A maximum of 7.5 or 9 hens per m² usable area is allowed, depending on the bodyweight of the hens (cut-off limit 2.4 kg). In multi-tiered aviaries, 20 hens per m² litter area or seven hens per m² available area are allowed. Laying hens in free-range production, including organic hens, are housed on litter in aviaries or single-tiered floor systems, and are allowed access to outdoor areas when weather conditions are favourable. The requirements for organic egg production in Sweden are set out by the Swedish certification body for organic production (KRAV^®), which meets the demands of the International Federation of Organic Agriculture Movements Basic Standards (IFOAM, 2005) and the European Union regulation for organic production (EEC 834/2007; European Commission, 2007). The stocking density indoors in organic production is six to seven hens per m² litter area depending on when the facility was built. According to KRAV^®, the available on-farm pasture area for organic hens is at least 4 m² per bird, and the maximum flock size in organic egg production is 3000 birds. In general, litter is not reused for consecutive laying hen flocks in Sweden, and “all-in-all-out” management is practiced by a majority of farmers at barn level, but not necessarily at farm level. Pullets (i.e. young laying hens) are brought to the farms at the approximate age of 15 to 16 weeks in order to acclimatize before start of lay, and birds intended for litter-based housing systems are usually reared in non-cage systems.

The major change in husbandry that awaits all European Union countries is likely to influence disease transmission, particularly of pathogens that are transmitted like ascarids (Blokhuis et al., 2005). The current situation regarding ascarid occurrence in European countries is not well known, although these infections in commercial laying hens have been reported in recent years from several countries, including Germany, Switzerland, Spain, the UK and Denmark, particularly from free-range flocks (Permin et al., 1999; Pennycott & Steel, 2001; Martín-Pacho et al., 2005; Kaufman & Gauly, 2009; Kaufmann & Hoop, 2009). Similarly, during the phase-out period of conventional battery cages in Sweden, ascarid infections soon became a regular finding at routine necropsies at the National Veterinary Institute (SVA), Uppsala, Sweden (unpublished observations). Further, adult A. galli are occasionally found in table eggs, which would not be acceptable to the public.

The objectives of the present study were to determine the prevalence and geographical distribution of ascarid infections in commercial Swedish laying hens, and to investigate associated risk factors. The occurrence of ascarid eggs in faeces was investigated both near the end of the nationwide change in housing systems in 2004, as well as 4 years later in 2008. Both surveys included the parasitological status of hens housed on litter and in cages, including furnished cages that to our knowledge have not been investigated previously. Also, the influence on parasitological results of farm and flock characteristics and management (e.g. cleaning and disinfection routines and basic biosecurity procedures) was investigated based on the findings and a questionnaire from 2004.

Materials and Methods

Target population and housing of hens

The target population consisted of commercial laying hens kept on farms listed in the national laying hen register of the National Board of Agriculture (SJV, Jönköping, Sweden). Registration is mandatory for all table egg-producing farms with at least 350 hens. The majority of hens in 2004 and almost all hens in 2008 were housed in furnished cages or in non-cage litter-based housing systems (Table 1).

Table 1.  Estimated number of laying hens and percentage of hens housed in different housing systems in 2004 and 2008.

	2004	2008
Number of laying hens (million)	5.6	5.8
Conventional cages (%)	13.5	0.7
Furnished cages (%)	24.8	40.2
Litter-based housing indoors^a (%)	55.0	50.8
Free-range, including organic hens (%)	6.7	8.3
^aTraditional single-tiered floor systems and multi-tiered aviaries.
Source: J. Yngvesson, Swedish Welfare Agency, personal communication, 2004; Association of Egg Producers in Sweden (2009).

Study design

All listed farms were invited by mail to participate in the study in the autumns of 2004 (n = 360) and 2008 (n = 340). The study design was cross-sectional. Invitations were issued by SVA in 2004, and jointly by the Association of Egg Producers in Sweden (SFS Svenska Ägg) and SVA in 2008. Non-responders were reminded once by mail. Sampling took place during August to December in 2004, and during October to December in 2008. In 2004, one flock on each farm was sampled. Random selection was not achievable because of lack of information on number of flocks on the farms. Therefore, a risk-based sampling strategy was used, where the oldest flock owned by the farmer, regardless of housing system and number of flocks, was sampled. The selection of the oldest flock was based on the following assumptions: once infected by ascarids, a flock would remain so during the entire production period because of continuous exposure to ascarid eggs and unavailability of anthelmintics (a registered anthelmintic for poultry became available in Sweden in 2009; Göransson, 2009); and recently infected flocks can only be expected to be identified by faecal egg count after the prepatent period, which in case of A. galli is at least 5 to 8 weeks and in H. gallinarum is 24 to 30 days (Permin & Hansen, 1998). Thus, false negative results would be minimized when the oldest flock on the farm was investigated. In 2008, the farmers were invited to submit faecal samples from all of their laying hen flocks provided that the birds had been transferred from the pullet-rearing farm at least 12 weeks prior to sampling; that is, exceeding the prepatent period of A. galli and H. gallinarum.

Sample collection and parasitological analyses

To investigate the suitability of various sampling sites in non-cage housing systems and the number of samples required to increase the probability to yield positive results from infected flocks, a pilot study was conducted in A. galli-infected flocks on three separate farms. Two of the flocks were housed in traditional single-tiered floor systems, including one organic flock, whereas the third flock was housed in a multi-tiered aviary. Twenty-six samples of at least 100 g faeces were obtained from each barn from litter, manure removal belts, from the manure scraper, manure auger, on and under slats, and from perches. On the organic farm, faeces were also collected from the outdoor run near the barn. The results showed that parasite eggs were present in all faecal samples obtained from the equipment inside the barn, in 22% of the litter samples, but in none of the samples collected from the outdoor run near the barn. Based on these results, farmers were instructed in both 2004 and 2008 on how to collect the samples depending on the system used. In litter-based housing systems, samples were obtained from manure removal belts, manure scraper, manure auger, perches, and on and under slats. In caged flocks, faeces were collected from manure removal belts. The farmers were encouraged to sample multiple sites and several manure belts in the house and to operate manure removal belts regularly during sampling. Each farmer collected four pooled samples per flock, each consisting of at least 100 g faeces, and the samples were sent overnight by surface mail to SVA. Each sample was thoroughly mixed and 3 g faeces per sample were examined individually by a modified flotation technique in a McMaster slide using a saturated NaCl solution with a minimum detection limit of 50 eggs per g faeces (Anonymous, 1986; Thienpoint et al., 1986). Attempts were not made to differentiate ova of Ascaridia spp. and Heterakis spp. Thus, in most infected flocks it was not known whether A. galli and/or H. gallinarum were present.

For parasite species identification in 2004, 16 laying hen flocks were randomly selected from among the flocks that shed parasite eggs. Five birds found dead by the farmer were collected from each flock and sent for necropsy to SVA. Eight of the flocks were housed in single-tiered floor systems or aviaries, and eight originated from organic farms. The gastrointestinal tract was removed and cut open. Intestinal contents were sieved through a 200 µm mesh sieve, and parasites were identified to species level (Permin & Hansen, 1998).

Questionnaire design

All participating farmers in 2004 were sent questionnaires that were returned together with the faecal samples. Full anonymity was guaranteed. Twenty-three questions related to general farm and flock characteristics, cleaning and disinfection routines between consecutive flocks, and application of biosecurity procedures were included in the questionnaire. A summary of the questionnaire items used in the statistical analysis is presented in Table 2. The questionnaire was closed or semi-closed, and it was tested for accuracy and relevance by five farmers prior to its use. Owing to lack of funding, the results of the questionnaire could not be validated by farm visits.

Table 2.  Summary of main items included in the questionnaire in 2004 to identify risk factors for ascarid infection on 186 Swedish laying hen farms.

Variable	Level	All farms	Ascarid-positive farms	Ascarid-negative farms
Farm characteristics
Number of units on farm	1: One	0.28	0.21	0.31
	2: More than one	0.55	0.63	0.52
	3: No information given	0.17	0.16	0.17
Housing system	1: Cages^a	0.23	0.02	0.29
	2: Single-tiered floor system	0.39	0.44	0.37
	3: Multi-tiered aviary system	0.19	0.14	0.21
	4: Free-range, including organic hens^b	0.19	0.40	0.13
	5: Unknown	0.005	0.00	0.01
Other housing system present	1: Yes	0.14	0.19	0.13
	2: No	0.84	0.77	0.87
	3: No information given	0.02	0.05	0.01
Poultry or game birds present on farm	1: Yes	0.09	0.09	0.08
	2: No	0.90	0.91	0.90
	3: No information given	0.02	0.00	0.02
Sampled flock characteristics
Rearing conditions	1: Cage rearing	0.26	0.14	0.29
	2: Reared on litter	0.63	0.84	0.57
	3: No information given	0.12	0.02	0.14
Hybrid (n = 183)	1: LSL/Lohman Brown/Silver	0.70	0.72	0.70
	2: Hy-Line White or Brown	0.26	0.23	0.27
	3: Other(s)	0.02	0.05	0.01
	4: No information given	0.02	0.00	0.02
Bird age (n = 173)		54 (43; 66)	52 (44.5; 68)	54 (43; 64)
Use of anthelmintics	1: Yes	0.005	0.02	0.00
	2: No	0.98	0.98	0.98
	3: No information given	0.02	0.00	0.02
House, equipment and management
Duration of egg production in barn	1: <1 year	0.16	0.07	0.18
	2: 1 to 5 years	0.29	0.23	0.31
	3: 6 to 10 years	0.18	0.35	0.13
	4: ≥11 years	0.35	0.35	0.36
	5: No information given	0.02	0.00	0.02
Age of housing equipment (n = 173)	1: ≤1 year	0.30	0.16	0.34
	2: 2 years	0.17	0.09	0.20
	3: 3 to 6 years	0.26	0.28	0.26
	4: ≥7 years	0.19	0.42	0.13
	5: No information given	0.07	0.05	0.08
Cleaning and disinfection
Cleaning between flocks	1: High-pressure cleaning	0.53	0.49	0.55
	2: High-pressure cleaning and steam washing	0.18	0.19	0.17
	3: Steam washing	0.10	0.16	0.08
	4: Dry cleaning	0.14	0.14	0.15
	5: No cleaning	0.01	0.02	0.01
	6: No information given	0.03	0.00	0.04
Disinfection interval	1: Between every flock	0.66	0.56	0.86
	2: Occasionally between flocks	0.05	0.12	0.03
	3: Never	0.21	0.23	0.28
	4: No information given	0.08	0.09	0.08
Biosecurity procedures
Hygiene barrier to house/unit	1: Yes	0.84	0.63	0.91
	2: No	0.14	0.37	0.07
	3: No information given	0.02	0.00	0.02
Change of footwear	1: Yes	0.84	0.72	0.87
	2: No	0.13	0.28	0.09
	3: No information given	0.03	0.00	0.03
Change of clothing	1: Yes	0.58	0.40	0.63
	2: No	0.39	0.58	0.34
	3: No information given	0.03	0.02	0.03
Continuous variables: median (first and third quartiles); categorical variables: proportions. Number of farms (n) contributing to each item is given if fewer than 186 farms contribute to a specific value. ^aCages = conventional battery cages n = 7 and furnished cages n = 37. ^bFree range flock n = 2, organic flock n = 38.

Statistical analysis

Comparisons between the two study years for number of ascarid-positive flocks, housing system, total number of flocks per participating farm, and age of sampled flock were performed using the chi-squared test and Fisher's exact tests. Following parasitological analysis in 2004, the identity of the farms was removed from the questionnaire form by the laboratory prior to the statistical analysis. The analytical unit of interest was the investigated flock of laying hens at a certain time point. Assuming similar epidemiology, the analysis was performed regardless of ascarid species. Data editing and all the statistical analyses were performed in Stata Software (Stata Statistical Software, release 11.0, 2009; StataCorp., College Station, Texas, USA). Associations between the dependent variable, flock positive for ascarid eggs, and each of 16 potential risk factors in the questionnaire were first investigated by univariable logistic regression analysis (Dohoo et al., 2010). Variables with P ≤ 0.20, provided that there was no collinearity (r < 0.70) between variables, were then considered for further analysis. Collinearity between variables was assessed pair-wise by calculation of Spearman rank correlations. Categories of categorical variables with too few observations were amalgamated when it was possible to make new categories based on biological or logical criteria. In other cases, such categorical variables were not used in the analysis or the category was omitted. A multivariable model was constructed using manual stepwise backwards regression analysis, where variables not significant in the model were re-entered whenever a new variable became significant, or a variable was removed. A variable was considered as a confounder if the point estimates of the coefficients in a model changed >20% with the potential confounder present. In the final model, a variable with P ≤ 0.05 was considered statistically significant and retained in the model. Biologically plausible interactions between the main effects were tested in the final model.

The fit of the model was evaluated with the Hosmer–Lemeshow goodness-of-fit test with the data partitioned into 10 deciles, and by visual examination of diagnostic plots (Hosmer & Lemeshow, 2000). Plots of Pearson residuals (r), leverage (h), delta beta (▵β), delta deviance (▵D), and delta chi-squared (▵χ²) versus the predicted values were constructed and evaluated. Observations with divergent values (i.e. –3 ≤ r ≥ 3, h > 0.3, ▵β > 1, ▵D > 4.0, or ▵χ² > 4.0) were considered outliers. Their impact was assessed by running the model without outliers, and comparing the coefficients between this model and the model using all observations.

Results

Prevalence and geographical distribution

Summarized data on the participating flocks are shown in Tables 2 and 3. In 2004, 200 of 360 contacted farmers (56%) sent faecal samples for parasitological analyses (i.e. 200 separate farms). Among the remaining farms, 32 (9%) had no hens on their farms at the time of the sampling, 40 (11%) did not wish to participate in the study, and 88 (24%) failed to respond. Based on available information in the national laying hen register of housing systems on all laying hen farms in Sweden, approximately equal proportions (2/3) of the four housing systems were represented in the study. The median number of hens per farm was lower on the sampled organic and free-range farms (median 3000 hens, range 500 to 17,100) compared with farms with cages (median 15,900 hens, range 1300 to 173,800) or litter-based systems indoors (median 9300 hens, range 350 to 289,000). In 2008, 330 laying hen flocks on 172 farms participated in the study (participation rate at farm level: 51%). When comparing the housing systems between years, there were significantly more flocks being housed in furnished cages and in multi-tiered aviary systems in 2008 than in 2004; that is, 27% and 23% for furnished cages and 27% and 19% for multi-tiered aviaries (P = 0.04) (Table 3). Moreover, there were significantly (P = 0.04) fewer flocks in single-tiered floor systems in 2008 than in 2004 (Table 3). These differences reflect the still ongoing changes in the laying hen industry structure in Sweden.

Table 3.  Occurrence of ascarid eggs in faecal samples from Swedish laying hen flocks in the autumn of 2004 and 2008.

Housing system	2004			2008^a
	Flocks (n)	Infected flocks (n)	Prevalence (%)	Flocks (n)	Infected flocks (n)	Prevalence (%)
Cages^b	42	1	2.4	46	2	4.3
Single-tiered floor system	72	20	27.8	42	12	28.6
Multi-tiered aviary system	36	6	16.7	46	24	52.2
Free-range, including organic hens^c	35	17	48.6	35	26	77.1
Unknown	1	0	0	0	0	0
Total	186	44	23.5	169	64	38.3
Data from the oldest flock (≥25 weeks of age) on each farm are included. ^aResults from four farms were excluded in 2008 because several flocks of the same age showed conflicting results. Ascarid eggs were observed in faeces from a younger flock on six farms where the oldest flock showed negative results. ^b2004: cages = conventional battery cages, n = 7; furnished cages, n = 37. 2008: enriched cages, n = 46. ^c2004: free-range flocks, n = 2; organic flocks, n = 38. 2008: free-range flocks, n = 2; organic flocks, n = 32.

The results of faecal egg count analyses in the oldest flock on the farms (hen age ≥ 25 weeks) in 2004 and 2008 are presented in Table 3. In order to obtain comparable datasets for both study years in terms of laying hen age and selection strategy, the results from 14 flocks/farms with hens younger than 25 weeks were excluded from the risk factor analysis for 2004. In 2008, nine flocks on four separate farms (of which one was a 24-week-old ascarid-infected flock) were excluded because the birds had been sampled before the stipulated 12 weeks spent in the barn (aged ≥ 25 weeks). Moreover, results from four farms in 2008 were excluded, because flocks of the same age on the same farm showed conflicting results. With these adjustments, the overall ascarid farm prevalence was 23% (n = 44/186) in 2004 and 38% (n = 64/169) in 2008. Thus, the farm prevalence was significantly higher in 2008 than in 2004 (P = 0.01). On six of the farms sampled in 2008, ascarid-infected younger hens were present, where the oldest flock on the farm was uninfected (Table 3). The arithmetic mean age for the flocks participating in 2004 was 54 weeks (range 25 to 90 weeks; n = 173), whereas it was 59 weeks (range 28 to 102 weeks; n = 167) in 2008, so the mean age differed significantly between years (P<0.05). Number of flocks per farm did not differ between years (P = 0.24). In addition to ascarid eggs, Capillaria spp. were observed in two organic flocks in 2004, and cestode eggs were detected in one organic flock in 2004. Parasite recoveries conducted from dead hens in 2004 showed that birds from all 16 randomly selected flocks among those that shed ascarid eggs also harboured adult worms. Among eight flocks housed on litter indoors, five had hens infected with A. galli and three with H. gallinarum. Four of eight organic flocks had hens with mixed infections, whereas two flocks were monospecifically infected with either A. galli or H. gallinarum.

Faecal samples were received from all over Sweden. The highest number of samples (n = 146 in 2004; n = 131 in 2008) originated from farms in the most southern region (Götaland), followed by the middle region (Svealand) (n = 42 in 2004; n = 26 in 2008), and samples from the northern region (Norrland) (n = 13 in 2004; n = 12 in 2008), which reflects the overall distribution of the laying hen population in Sweden. The ascarid prevalence at farm level varied between regions both in 2004 (Götaland: 26%, Svealand: 17%, Norrland: 15%) and in 2008 (Götaland: 35%, Svealand: 58%, Norrland: 33%), but there were no significant regional differences within years. There was a significant increase in prevalence in the region of Svealand from 2004 to 2008 (P = 0.001). In total, 90 farmers submitted faecal samples from at least one flock in both 2004 and 2008, of which 11 farms (12%) were infected and 51 (57%) were not infected on both occasions. Among 75 farms that were uninfected in 2004, 24 (32%) had become infected by 2008, which indicates an incidence of around 7%. Four (27%) of the 15 farms infected in 2004 showed a negative result in 2008. One of these was a multi-unit and multi-age farm with single-tiered floor systems. This farm had changed ownership between the two sampling occasions, and the new owner introduced strict biosecurity procedures, and all barns were thoroughly cleaned and disinfected during an extended down-time period. A second farm was kept empty of hens for a long period of time, while all equipment was changed from conventional cages to a multi-tiered aviary system. The flocks on the remaining two farms came from the same barns with hens housed in a single-tiered floor system or organic production on both sampling occasions. The sampled flocks in 2008 were among the youngest sampled flocks (28 and 33 weeks of age, respectively).

Questionnaire

The questionnaire in 2004 was returned by all 200 farmers, of which 186 met the inclusion criteria (flock age ≥ 25 weeks). The results of the questionnaire are summarized in Table 2. The farmers were asked whether they had seen any ascarid parasites in faeces or whether they had received complaints from the slaughterhouse, egg-packing plant or from table egg consumers, and 19 farmers answered yes (10%). Seventy-four per cent of these farmers had flocks with ascarid infection in the present study. Five farmers answered that they had previously tried to eliminate environmental ascarid egg contamination on their farm. Only one farmer of the 186 participating stated that he/she had used anthelmintics (in an earlier flock).

Univariable and multivariable analysis

A total of 21 variables were screened in the univariable analysis, and nine of those presented P ≤ 0.20. Univariable analysis of associations between use of disinfectants and ascarid infection was not performed, because 22 different products or combinations of two or more products had been applied by the farmers, and nine farmers were unaware of the identity of the product they had used. Also, most of the applied products lacked a documented effect against ascarid eggs. Among the 43 ascarid-infected flocks, nine different products had been applied, of which only one had a documented effect against ascarid ova (used by one farm). Moreover, collinearity was detected between the rearing system for the pullets and housing system on the laying hen farms, as well as between the manure removal system and housing system. Housing system gave the best model fit of these three variables and was the one eligible for the multivariable analysis. Of six variables offered to the multivariable logistic regression model (i.e. age of housing equipment, type of housing system, hygiene barrier to house/unit, duration of egg production in barn, change of footwear, change of clothing), three remained with P < 0.05 (Table 4). The variable associated with an increased risk of having a flock positive for ascarid eggs was to have housing equipment ≥7 years old in the housing system where the flock was housed; the odds ratio (OR) for >7 years versus <1 year was 7.5 (95% confidence interval [CI] = 2.3 to 25). Variables associated with having a flock negative for ascarid ova were to have the hens in cages, and to have a hygiene barrier at the house/units. The OR of no hygiene barrier compared with a barrier was 7.7 (95% CI = 2.5 to 24).

Table 4.  Final multivariable logistic regression analysis of variables significantly associated with laying hen flocks with or without ascarid eggs in faecal samples.

Variable	β	SE(β)	OR	95% CI (OR)	P value
Intercept	–1.40	0.84	–	–	–
Age of housing equipment
1: ≥7 years	Ref.	–	–	–	–
2: 3 to 6 years	–1.46	0.57	0.23	0.08; 0.71	0.01
3: 2 years	–1.36	0.68	0.26	0.07; 0.97	0.05
4: ≤1 year	–2.02	0.61	0.13	0.04; 0.44	0.001
Housing system
1: Free-range/organic	Ref.	–	–	–	–
2: Multi-tiered aviary system	–0.93	0.65	0.39	0.11, 1.42	0.15
3: Single-tiered floor system	–0.97	0.51	0.38	0.14, 1.03	0.06
4: Cages	–3.45	1.15	0.03	0.003, 0.30	0.003
Hygiene barrier at houses/units
1: Yes	Ref.	–	–	–	–
2: No	2.04	0.58	7.72	2.48, 24.1	<0.001
Significance, P ≤ 0.05. n = 170, pseudo-R ^2 = 0.28; Sweden, 2004. β, regression coefficient; SE, standard error; Ref., reference.

Model fit

The multivariable model showed a good fit; the Hosmer–Lemeshow χ² (eight degrees of freedom) was 5.13 (P = 0.74). When looking at different plots of r, h, ▵β, ▵D, and ▵χ², several divergent observations were seen depending on which diagnostic value was addressed. A total of 101 observations had h > 0.3 (0.35 to 0.67), 30 observations had ▵β > 1 (1.3 to 2.9), and two observations had ▵D > 4.0 (4.1 for both), but the coefficients did not change considerably and the model did not improve much with deletion of the divergent observations, so all observations were kept.

Discussion

Few data are available on flock prevalence of ascarid infections from egg-laying chickens in industrialized countries (Permin et al., 1999; Pennycott & Steel, 2001; Martín-Pacho et al., 2005; Kaufman & Gauly, 2009; Kaufmann & Hoop, 2009). Still, the findings in these studies jointly point to an increased risk of infection in laying hens housed on litter where there is a higher level of faecal contact, and in particular in free-range egg production. Our results are in close agreement with the earlier studies, and they also clearly show that ascarids have increased considerably in Swedish laying hen flocks since the ban on conventional battery cages in 1999. However, no significant difference in farm prevalence was found between hens kept on litter indoors and those that had access to pens and/or pasture. On the other hand, hens kept in cages, including furnished cages, were rarely infected in this study, probably because of the more limited faecal exposure.

The results from the surveys in 2004 and 2008 were based on datasets collected according to slightly different selection strategies (i.e. only the oldest flock in 2004 versus between one and six flocks in 2008), whereas sample collection and parasitological analyses were performed in the same way in both years. Still it can be argued that this may have impacted the observed increase in ascarid prevalence between 2004 and 2008. However, potential selection bias was dealt with as follows: by only including data from the oldest flock in 2008; by omitting four farms in 2008 where there were conflicting results between the oldest flocks on the same farm (when several flocks of the same age were present); and by including only flocks aged ≥25 weeks in both study years. By these measures, we are convinced that the increase in ascarid prevalence reflects the true situation. This is supported by the fact that as many as 32% of the farms that were uninfected in 2004 had become infected 4 years later. Thus, during the past decade there has been a rapid spread of ascarids in commercial laying hens in Sweden. Interestingly, this was so irrespective of whether the flocks were managed according to the national regulations for organic production or not. According to our results, the risk of being infected increased along with the changed housing conditions. We also noticed that the risk increased with the age of equipment in the barn, indicating that for unknown reasons it may take some years before the farms become infected.

How ascarids are transmitted to chickens on laying hen farms in Sweden is not known. Hypothetically, ascarids may be introduced by infected wild or domestic birds and/or by wild animals, people or equipment acting as mechanical vectors for the parasite eggs. Once introduced on a farm, ascarids are likely to spread between barns on multi-unit and multi-age farms. Furthermore, ascarids will normally remain present over time, as they are highly resilient to variations in humidity, temperature and pH, and therefore are likely to survive for long periods in the environment on the farms (Permin & Hansen, 1998). Although ascarids in pullets cannot be ruled out as an important source of infection, Swedish pullets have not as yet, to the authors’ knowledge, been found infected with A. galli during the rearing period or upon arrival at the egg production units (unpublished observations). However, the number of pullet flocks that have been investigated until now remains low. Thus, further investigation is needed before this possibility can be ruled out. Another source of ascarid transmission is from other poultry on the same farm since the “all-in-all-out” management strategy may only be applied at barn level on multi-unit farms. However, other poultry and game birds were only found on a subset of the farms investigated and the analysis did not indicate that they were a risk factor (Table 2). It has also been shown that certain free-living wild birds may be infected with A. galli and/or H. gallinarum (Mozogovoi, 1953, translated 1968). Thus, these can be regarded as a possible infection source. However, in this study the statistical analysis failed to detect a significant difference in prevalence between farms that kept their hens in non-cage systems either only indoors or both indoors and outdoors, which would be an expected finding if wild birds were a primary source. A third possible infection source consists of farm-to-farm mechanical transmission of parasite eggs by contaminated people and/or equipment. Despite laying hen farms often being geographically isolated, they have many visitors such as egg transporters, farm workers, housing and feed consultants, workmen, inspectors, veterinarians, pest controllers, and occasionally, buyers of eggs and end-of-lay hens. Furthermore, transport crates for pullets and end-of-lay hens are often transported between farms, and egg trays travel between the farm and the egg-packing plant. Contamination of these materials by ascarid eggs is probably difficult to avoid with the sanitary measures that are applied today. The same applies to equipment used for cleaning and disinfection, which is sometimes moved both between units on the same and/or between different farms. Similarly, personnel and vehicles transporting birds, feed and equipment can also carry the infection.

Biosecurity measures on laying hen farms consist of a set of management practices, implemented throughout the operation, such as barriers to the external environment. Presence of entry rooms, hygiene barriers with washing facilities, protective clothing and footwear both for farm workers and visitors, together with cleaning and disinfection routines as well as on-farm carcass disposal techniques, are in general imperative. In this study, an attempt was made to identify factors associated with an increased risk of hens being infected with ascarid eggs. Notably, the absence of a hygiene barrier (defined as a physical partition, e.g. a bench or a wooden board at the entrance to the poultry barn or unit) was highly associated with an increased risk of ascarid infection (OR = 7.7; 95% CI = 2.5 to 24). This suggests that ascarids are probably often transmitted horizontally between different farms. However, to obtain a dataset on biosecurity measures by way of a questionnaire study is difficult. First, farmers may not always fully comply with their own biosecurity routines as presented in a questionnaire. Secondly, the questionnaire was not tested for repeatability or accuracy by visiting the farms. Thirdly, it needs to be realized that the infection status we observed was often based on a once-only snapshot investigation. Our results also sometimes showed that all flocks from the same farm did not always show the same infection status. Still, the outcome of the questionnaire study (Table 2) was consistent with the actual situation based on our long-term experience and structural knowledge based on prior farm visits and poultry diagnostics. Although modern biosecurity procedures have been applied on many farms included in the present study, specifically designed measures to avoid the introduction of ascarids have rarely been applied. Nevertheless, we suggest that strict hygiene barriers are recommended for laying hens both to improve the biosecurity of the farm in general and to lower the risk for introduction of ascarids in particular. Hypothesis-driven studies are clearly required to elucidate how ascarids are transmitted between geographically separated commercial laying hen farms.

Persistent environmental contamination by ascarid eggs between consecutive flocks is difficult to avoid given their resilient nature (Permin & Hansen, 1998). With few exceptions, a majority of the farms investigated in both surveys remained infected during the course of the study. Routine cleaning and disinfection between flocks do not appear to protect pullets from being infected upon arrival at the egg production units. Also, flubendazole, which is the only available anthelmintic for poultry in Sweden (since 2009) offers only short-acting effect, and the use of anthelmintics is not a realistic solution on organic farms. To overcome these difficulties, measures that will prevent further spread of ascarid eggs must be addressed in the future. The same applies to run management strategies for free-range and organic laying hens as highlighted by Heckendorn et al. (2009). Given the fact that there is at present little to offer the producers in terms of prevention, and in particular efficient elimination of parasite eggs once an ascarid infection has become established, it is important to find out how we can avoid introduction of ascarids to new farms.

Acknowledgements

The Swedish Board of Agriculture (SJV) provided financial support. Birgitta Andersson, Kenneth Backström, Bodil Christensson and Susanne Johansson are thanked for technical assistance and Ann-Charlotte Andersson for secretarial services. The authors would especially like to thank all participating farmers for their cooperation.