Environmental tolerance of free-living stages of the poultry roundworm Ascaridia galli

Veterinary Parasitology 209 (2015) 101–107

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Environmental tolerance of free-living stages of the poultry roundworm Ascaridia galli Behdad Tarbiat a , Désirée S. Jansson b , Johan Höglund a,∗ a Department of Biomedical Sciences and Veterinary Public Health, Section for Parasitology, Swedish University of Agricultural Sciences (SLU), P.O. Box 7009, SE-750 07 Uppsala, Sweden b Department of Animal Health and Antimicrobial Strategies, National Veterinary Institute (SVA), SE-751 89 Uppsala, Sweden

a r t i c l e

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Article history: Received 9 July 2014 Received in revised form 15 January 2015 Accepted 25 January 2015 Keywords: Ascaridia galli Embryonation Chlorocresol Environmental conditions L3 Parasite egg

a b s t r a c t The poultry roundworm Ascaridia galli is re-emerging in laying hens in many European countries due to the increase in non-caged housing. A series of in vitro experiments was carried out to study the in ovo larval development (embryonation) under different environmental conditions. Between 83% and 96% of the eggs developed to L3 within 7–21 days of incubation in water between 20 and 30 ◦ C. Twenty-six percent completed development at 33 ◦ C and 4% at 35 ◦ C after 31 days. At 15 ◦ C parasite egg development was low with 8% L3 after 56 days. In another trial larval development occurred, when parasite eggs were exposed to freeze–thaw cycle (30 to 12 h) followed by incubation for 2 weeks at 25 ◦ C. Alkaline and acidic conditions in the range of pH 2.5–12.5 had no adverse effect on development. Oxygen and relative humidity above 70% were necessary for development to occur. Thus, some A. galli eggs may complete development at conditions prevailing in poultry barns in temperate climate zones throughout the year. Although exposure to a 1% or 2% dilution of the broad-spectrum disinfectant chlorocresol for 4 h or longer was ovicidal, further work is required to improve the method of application in the field. © 2015 Elsevier B.V. All rights reserved.

1. Introduction The poultry roundworm Ascaridia galli (syn. Ascaridia granulosa, Ascaridia lineata, Ascaridia perspicillum) is a nematode found mainly in the small intestine of galliform birds. It is re-emerging today in modern facilities for egg production. Although the parasite life cycle is direct, to reach the infective stage, the eggs must undergo a period of embryonation in the external environment (Permin and Hansen, 1998). According to researches the length of this period takes between one and three weeks and is

∗ Corresponding author. Tel.: +46 18 671000; fax: +46 18 309162. E-mail address: [email protected] (J. Höglund). http://dx.doi.org/10.1016/j.vetpar.2015.01.024 0304-4017/© 2015 Elsevier B.V. All rights reserved.

dependent mainly on oxygen (Ackert, 1931), temperature, and humidity (Reid, 1960; Permin and Hansen, 1998). Chicken egg production in litter-based and free-range housing systems has increased in Europe after the implementation of the EU-wide ban on conventional battery cages for laying hens (Directive 99/74/EC). These recent housing changes have highlighted the need for improved control of infectious agents, such as A. galli, which rely on a faecal–oral transmission route. Despite an increased awareness, A. galli has become more prevalent in noncaged laying hens in many European countries and it is again of concern to the egg industry (Permin et al., 1999; Jansson et al., 2010; Höglund and Jansson, 2011; Kaufmann et al., 2011; Sherwin et al., 2013). Although management practices have been shown to be efficient with regard to parasite control in enriched cages (Jansson et al., 2010),

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debate continues about sustainable strategies for parasite prevention and control in non-caged housing systems. Many studies have been carried out on the biology of A. galli. Although some attention has been paid to its free-living stages, most of our knowledge about the embryonation or in ovo larval development in A. galli is based on American studies carried out between 1930 and the late 1960s (Ackert, 1931; Ackert and Cauthen, 1931; Levine, 1937; Roberts, 1937; Christenson et al., 1942; Ackert et al., 1947; Reid, 1960; Tongson, 1967). As external stages of parasites may adapt to local conditions, it is questionable whether dated results still apply to European conditions. While, there are some recent studies of the free-living stages of related nematodes conducted under field and laboratory conditions (e.g. Gaasenbeek and Borgsteede, 1998; Saunders et al., 2000), the results are limited and further research is needed. The aim of this project was to investigate how environmental factors, such as temperature, pH, humidity and oxygen, influence the development and survival of A. galli eggs under laboratory conditions. In addition, we also tested the effects of two dilutions (1% and 2%) of the disinfectant, chlorocresol, on egg viability in vitro. A series of laboratory experiments was conducted to study the intra capsular development of A. galli under laboratory conditions to confirm, if possible, and extend our knowledge about this parasitic species. It was hypothesized that A. galli eggs may have undergone adaptation to their environmental condition such as relatively constant daily mean temperature in poultry houses. Hypothetically, data from this study may assist in developing more efficient control measures for A. galli. 2. Material and methods 2.1. Preparation of parasite eggs Parasite egg suspensions were prepared from fresh faecal material collected in the same manner from the manure belts on two occasions on farms in Sweden with commercial laying hens. Both farms, following necropsies and parasitological analyses on numerous occasions had been shown to be naturally mono-infected with A. galli. To obtain clean parasite eggs, faeces were mixed with cold tap water and poured progressively through 600 and 150 ␮m aperture metal sieves. The eggs were then concentrated in Visser sieve funnels (110 and 70 ␮m) and collected by flotation in saturated NaCl (SG 1.18–1.20) in 50 ml screw-cap tubes centrifuged (IES CL50 centrifuge, Techtum Lab AB® ) at 425 × g for 5 min. Finally the eggs were washed in tap water and stored at 4 ◦ C without antifungal or antibacterial agents and used within 4 weeks. The species identity of the parasite eggs was confirmed at the start of the trial by an in-house PCR (data not shown). The first batch of eggs was used in the temperature trials and the second batch for the rest of the trials. 2.2. Egg developmental stages The parasite eggs were classified microscopically into four developmental stages (Fig. 1); unembryonated (UM),

developing early or late morula (EM/LM), vermiform containing the L1 /L2 stages (V), or embryonated (L3 ). Eggs with an abnormal intra-capsular mass or disrupted eggshell were considered dead (D), were either dark or completely translucent, and possessed a shrunken internal embryonic mass or had lost its integrity (Fig. 1A). Unembryonated eggs contained a single cell, which almost completely filled the eggshell and appeared granulated (Fig. 1B). Eggs undergoing mitosis were classified as the EM stage if they contained 2–16 cells (Fig. 1C1 ) or as LM if equipped with >16 cells without signs of differentiation (Fig. 1C2 ). The V stage was characterized by differentiation into a nonmotile (tadpole-like) embryo, which would further develop into a vermiform stage that would almost fill the entire capsular space and have a higher terminal opacity (Fig. 1D). Eggs that had completed development contained a coiled slender motile larva (L3 ) which was longer than V-stage larvae and had a thinner cylindrical body with less terminal opacity (Fig. 1E). Because classification of different stages was done solely based on microscopic observation, specific morphological changes associated with intra-capsular moulting or retention of the larval sheath was not observed. Thus, embryonated eggs in this study were assumed to be in the L3 stage and infective. Developmental stages were investigated and documented by an Olympus BX40 microscope equipped with a digital Olympus DP50 camera. 2.3. Temperature trials In trials 1 and 2, flat-bottomed 50 ml tissue culture flasks (Falcon) were prepared with ∼2800 UM eggs in 10 ml water and incubated in climatic cabinets (Nüve ES110) at 5, 10, 15, 20, 25, 30, 33 and 35 ◦ C. The eggs had been stored in tap water for 4–7 days at 4 ◦ C were used in both trials and they were examined once a week for 42–56 days at 5–30 ◦ C (trial 1), and after 9, 16 and 31 days of incubation at 33 and 35 ◦ C (trial 2). Due to evaporation loss, the water volume was adjusted by the addition of distilled water as required. A minimum of 100 eggs was observed at each temperature and time point and the percentage of egg in each developmental stage were determined microscopically as described above (Fig. 1). In trial 3, a climatic cabinet was programmed to simulate a temperature decrease (3 ◦ C/h), starting at 20 ◦ C until the temperature reached 5 ◦ C (maintained for 5 h), followed by a further decrease (3 ◦ C/h) until it reached −5 ◦ C (maintained for 24 h). At the start of the experiment 12 Eppendorf tubes each containing ∼600 ␮l of egg suspension (∼7 eggs/␮l) were incubated. When the cabinet reached −5 ◦ C, tubes in duplicates were moved after 30 min, 60 min, 3 h, 6 h and 12 h to another incubator at 25 ◦ C and each tube was incubated for two additional weeks. 2.4. Relative humidity Approximately ∼200 eggs in 30 ␮l egg suspension were pipetted on 20 microscope slides and air dried for 5 min. The RH levels of 50%, 70% and 90% were created using mixtures of water and glycerol in compartmentalized disposable Petri dishes (Fisherbrand® ) (Table 1). In each Petri dish one microscope slide was placed above the

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Fig. 1. Classification of different developmental stages of Ascaridia galli eggs utilised in the present study.

water/glycerol mixtures, sealed with Parafilm® and incubated at 25 ◦ C. In addition, eggs incubated in distilled water served as control (100% RH). The RH in the dishes was measured using a hygrometer (Testo 605-H1TM ) prior to the experiment. Petri dishes were removed at 4-day intervals up to day 16, and in ovo larval development was investigated as described earlier.

different buffer solutions (Table 2) for 2 weeks at 25 ◦ C. All buffers had a molarity of 0.1 M and were passed through a 0.2 ␮m filter prior to use. Distilled water was used as a control medium. Two 24-well tissue culture plates (Nunc® ) were prepared and 30 ␮l of egg suspension (∼200 eggs plus 1970 ␮l of the buffers or water) were added to wells in triplicates. Plates were sealed with Parafilm® , and incubated

2.5. Environmental pH

Table 2 Biological buffer solutions used for pH experiment.

To determine the influence of environmental pH values on in ovo larval development, A. galli eggs were incubated in Table 1 Relative humidity (RH) of water/glycerol mixtures at 25 ◦ C used for the RH experiment in this study. RH (%)

50 70 90 100

Mixture of glycerol/water Glycerol (ml)

Water (ml)

79 64 33 0

21 36 67 100

pH ranges

Buffera

2.5 3.5 4.5 5.5 6.5 7.5 8.5 9.5 10.5 11.5 12.5

C2 H5 NO2(0.75 g) /HCl, 1 M(10 ml) C6 H8 O7(2.10 g) /NaH2 C6 H5 O7(2.94 g) C6 H8 O7(2.10 g) /NaH2 C6 H5 O7((2.94 g) C6 H8 O7(2.10 g) /NaH2 C6 H5 O7(2.94 g) KH2 PO4(1.36 g) /NaOH, 1 M(10 ml) KH2 PO4(1.36 g) /NaOH, 1 M(10 ml) NH2 C(CH2 OH)3(1.21 g) /HCl, 1 M(10 ml) C2 H5 NO2(0.75 g) /NaOH, 1 M(10 ml) C2 H5 NO2(0.75 g) /NaOH, 1 M(10 ml) HNa2 O4 P(0.84 g) /NaOH, 1 M(10 ml) KCl(0.75 g) /NaOH, 1 M(10 ml)

Amount of each substance solved in 100 ml distilled water. a Desirable pH was adjusted by titration.

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until the eggs in the control group developed into L3 14 days later. The in ovo larval development was examined with a mean number of 150 ± 18 parasite eggs per pH condition. 2.6. Hypoxia AnaeroGen sachets (Oxoid Ltd.) were used according to the manufacturer’s instructions to investigate the effect of hypoxia (≤1% O2 ) on in ovo larval development. Two groups, one with UM eggs and another with pre-incubated developed eggs containing V or L3 , suspended in water were set up in 24-well tissue culture plates in triplicates. The plates were incubated in an anaerobic plastic pouches (Oxoid Ltd.), whereas the controls had access to atmospheric oxygen. All plates were incubated at 25 ◦ C for 2 weeks, with one plate from each group removed every 4 days to check for any possible egg development (Fig. 1) or larval motility by microscopic examination. The same plates from the anaerobic treatment were then incubated aerobically at 25 ◦ C for another 2 weeks before final examination. 2.7. Disinfectant The ovicidal efficacy of the broad-spectrum disinfectant chlorocresol (Interkokask, InterHygiene® GmbH) against A. galli eggs was investigated. Samples of ∼100 ␮l egg suspension with ∼700 UM eggs were placed in 50 ml Falcon tubes in triplicates, and were then exposed in 10 ml of 1% or 2% (v/v%) solutions of chlorocresol. Following 4, 24 and 96 h of incubation the eggs were washed four times in distilled water by centrifugation for 3 min at 425 × g. Following incubation for 2 weeks at 25 ◦ C, at least 150 eggs per sample were examined per concentration. As the temperature trial was run simultaneously, it served as a control for the disinfectant trial. 2.8. Statistical analyses Data were summarized in Excel® (Microsoft® Mac version 14.2.2), and exported to JMPTM version 10.0 (SAS Institute Inc. Cary, NC, USA) and/or GraphPad Prism® version 6.00 (GraphPad Software, La Jolla, CA, USA), where statistical analyses and graphical illustrations were carried out. Differences in results from the temperature trials were tested with the Chi-square and Fisher’s exact tests in contingency tables. The replicated data from trial 3 were analysed by a generalized linear model (GLM) with several different assumptions to test the robustness of the model. The proportion of eggs with L3 was the dependent variable, whereas temperature conditions and time were independent factors. The significance level was set to p < 0.05. 3. Results 3.1. Temperature experiments The outcome of the in ovo larval development at constant temperatures in the range of 5–35 ◦ C (trials 1 and 2) are shown in Fig. 2. Between 110 and 279 eggs were examined at each temperature and time point indicating

Table 3 Percentage in ovo larval developmental stages of Ascaridia galli eggs maintained under different relative humidities (RH) at 25 ◦ C. RH (%)

UM

EM/LM

V

Day 4 50 70 90 100

L3

D

n

1 6 4 4

10 94 1 1

0 0 95 95

0 0 0 0

89 0 0 0

221 209 196 200

Day 8 50 70 90 100

0 4 5 <1

0 96 0 7

0 0 95 93

0 0 0 0

100 0 0 0

243 201 212 187

Day 12 50 70 90 100

0 0 3 4

0 4 0 0

0 1 97 9

0 0 0 87

100 95 0 0

206 215 205 201

Day 16 50 70 90 100

0 0 3 2

0 0 0 0

0 0 0 0

0 0 97 98

100 100 0 0

217 234 230 189

UM, unembryonated; EM, early morula; LM, late morula; V, vermiform; L3 , infective larvae; D, dead; n, number of A. galli eggs examined.

a significant increase in the proportion of eggs developing with increasing temperature in the interval 15–30 ◦ C (2 = 114.3–217.9; p < 0.001). At 5 ◦ C only 0.5% of the eggs were in the V stage on days 14 and at 10 ◦ C 2.5% of the eggs reached the V stage on day 21. Due to the difficulty of selecting solely undeveloped eggs at the start of the trial, the minor proportion was in the developing stage at the start of the experiment was included in the overall development rate in Fig. 2. Despite the slow development rate at 15 ◦ C the V stage was observed in 90% of the eggs at 42 days, whereas the L3 stage appeared in 8% of the eggs on day 56. At 20, 25 and 30 ◦ C L3 appeared on days 21, 14 and 7, respectively. At 33 and 35 ◦ C, development of the eggs was markedly decreased as 26% and 4% embryonated eggs counted on day 31. The majority of the eggs (n = 172 ± 9.2) in trial 3, which were exposed to temperatures below 0 ◦ C for up to 24 h developed without freezing into L3 after subsequent incubation at 25 ◦ C for two additional weeks. The following is the development rates as the exposure time decreased: 82.5% ±2.1 after 12 h of exposure, 80% ±2.8 at 6 h, 80% ±2.5 at 3 h, 80% ±2.8 after 1 h and 83% ±1.4 at 30 min exposure. The control groups showed 85% ±0.8 eggs at L3 . There was no significant difference between the groups (p < 0.05). 3.2. Relative humidity Between 199 ± 12 and 222 ± 16 eggs were examined per condition. At 100% RH, 87% of the parasite eggs were fully developed on day 12 and 98% on day 16. However, at 50% RH, signs of embryonation were observed in some eggs on day 4, whereas 100% were dead on day 8 (Table 3). At 70% RH, the majority of the eggs started to develop within 8 days, but most were dead on day 12. When exposed to 90% RH, ≥95% of the eggs were in the V stage between days 4 and 12 and had developed to V or L3 on day 16.

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Fig. 2. Percentages of different in ovo larval developmental stages of Ascaridia galli at different constant temperatures in the range of 5–35 ◦ C. Note that the data in trial 2 were examined after 9, 16 and 31 days only.

3.3. pH Of the eggs in the control group 84% were fully developed within 14 days. Mean L3 development varied between

77% and 84% in each pH concentration. As no significant reduction in development was observed in relation to the pH range 2.5–12.5, no optimal pH for egg development could be determined.

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3.4. Hypoxia No signs of in ovo larval development were observed in a mean number of 236 ± 5 parasite eggs during exposure to hypoxic conditions between 4 and 16 days at 25 ◦ C. When the eggs were incubated aerobically for two additional weeks, 87–90% developed to L3 . Morphologically, eggs (212 ± 7) containing L3 larvae appeared normal but no sign of motility was observed. 3.5. Chlorocresol disinfectant Chlorocresol dilutions of 1% and 2% were both effective irrespective of the time of exposure. All eggs examined showed degenerative changes of the intra-capsular cell mass. 4. Discussion Commercial egg production of today relies on biosecurity to protect hens from pathogens such as the free-living stages of parasites. To prevent, mitigate and control infections it is imperative to understand the biology of the microorganisms involved. This study was carried out to provide information on the influence of environmental conditions on the in ovo larval development of A. galli eggs, including some novel aspects. Infectivity and viability of larvae were not investigated. In trials 1 and 2, development was enhanced with increased temperatures in the interval 20–30 ◦ C with L3 appearing after 7–21 days of incubation (Fig. 2). This is in close agreement with earlier studies on A. galli (Ackert, 1931; Christenson et al., 1942; Reid, 1960; Tongson, 1967) and also with the related pig roundworm Ascaris suum (Arene, 1986). At 15 ◦ C, development was slow but more than 90% of the eggs eventually developed to the V stage at 42 days of incubation, and 8% had reached L3 at 56 days. This suggests that development may take place from around 15 ◦ C, which differs from previous studies where the minimum temperature at which the development occurred was 17 ◦ C (Reid, 1960; Tongson, 1967) Thus, some in ovo larval development is likely to occur in poultry barns throughout the year even in a temperate climate such as in Sweden, where temperatures at the floor level may fluctuate around 15 ◦ C during winter (Höglund and Jansson, 2011). Although the conditions for egg development was near optimal at 25 and 30 ◦ C, development was arrested at 35 ◦ C for many eggs at the UM/LM stage. Still, 4% reached L3 by day 31, which differs from earlier studies where no development was observed at this temperature (Roberts, 1937; Reid, 1960; Tongson, 1967). Despite the significant adverse effect of the temperature above 33 ◦ C on the development of the parasite eggs, results from a previous study on A. galli have shown that high temperatures (steam cleaning or hot water high pressure cleaning) did not prevent re-infection of the consecutive flock in the same barn (Höglund and Jansson, 2011). Furthermore, short-term exposure (up to 24 h) of fresh UM eggs to sub-zero temperatures (trial 3) had only minor effects on in ovo larval development following subsequent incubation at 25 ◦ C for 2 weeks. This is consistent with

earlier findings (Levine, 1937; Roberts, 1937) and may suggest a strategy for cold tolerance in A. galli in accordance with other nematodes (Wharton and Allan, 1989), even if the mechanism in A. galli is still unknown. There is limited information on these aspects in A. galli (e.g. Ackert and Cauthen, 1931). Further work needs to be done to examine the effect of prolonged exposure to sub-zero temperatures and repeated freeze–thaw cycles on eggs in different stages of development. To our knowledge, there are two previous papers on the moisture requirements for embryonation in A. galli eggs (McRae, 1935; Hansen et al., 1953). Together with earlier studies, our results suggest that approximately 85–90% RH or more is required for embryonation of A. galli eggs at the optimal temperature of 25 ◦ C (Table 3). Thus, our findings confirm the susceptibility of ascarid eggs to desiccation described in Gaasenbeek and Borgsteede (1998). Still, A. galli eggs might be able to develop at a temperature around 30 ◦ C and 70% atmospheric humidity in about 7 days. However, it remains unknown whether A. galli can withstand repeated dehydration/rehydration cycles, and if the permeability of the eggshell is modified to control for water loss in response to desiccation. Although, knowledge about variations in the microclimate in poultry barns and in the outdoor environment is limited, it is believed that infective hotspots are generated in certain places where conditions are favourable. Nematode eggs are in general considered to be highly resistant to different pH values due to the presence of a complex eggshell with an inner lipid layer that protects the developing embryo, particularly in ascarids (Bird and Bird, 1991). In the pH trial, the mean percentage of in ovo larval development was higher than 78% in the pH range 2.5–12.5, which confirms the high tolerance of A. galli eggs to extreme pH levels. Although neutral pH conditions are most likely to be found in the external environment, the eggs must survive gastric passage with a pH of 2.5–4.8 in the chicken proventriculus and gizzard (Denbow, 2000). Nevertheless, it has been shown that preservation of A. galli eggs in an acidic medium was detrimental to the establishment of infection in chickens (Lazdina and Grinberga, 1978). Similarly, the combination of high temperatures and aqueous ammonia inactivated A. suum eggs in toilet waste (Nordin et al., 2009). It has been demonstrated previously for ascarids including H. gallinarum (Saunders et al., 2000) and A. suum (Gaasenbeek and Borgsteede, 1998) that periods of hypoxia will slow egg development. Similarly, in our study A. galli eggs were inhibited by anaerobic conditions, while most eggs remained viable and resumed development in response to oxygen. However, the long-term effects of hypoxia need to be further studied. The broad-spectrum disinfectant chlorocresol (Interkokask, InterHygiene® GmbH) is one of the few products available today, claimed to be effective against ascarid eggs. Chlorocresol, as it was used in our study proved to be ovicidal in vitro even at a substandard concentration. However, for unknown reasons chlorocresol seemed not to be fully effective against A. galli eggs in poultry barns (Höglund and Jansson, 2011). Ascarid eggs are thick-shelled and have an outer sticky coat (Bird and

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Bird, 1991), by which they can adhere to surfaces and equipment in poultry barns. In this way, A. galli eggs may escape exposure if disinfectant does not reach all cracks and crevices in poultry houses. Furthermore, the corrosive nature of chlorocresol prevents effective usage on equipment. Obviously, A. galli eggs are exposed to a set of more complex environmental condition in the field than in the laboratory. Overall, the results from the present study suggest that A. galli eggs develop at a broader constant temperature range (15–35 ◦ C) than previously reported. Although a high relative humidity and aerobic conditions are necessary, conditions for in ovo development to L3 are likely met year around in poultry barns in temperate climates. Financial support This work was supported by the research programme SLU EkoForsk and The Swedish Research Council Formas. References Ackert, J.E., 1931. The morphology and life history of the fowl nematode Ascaridia lineata (Schneider). Parasitology 23, 360–379. Ackert, J.E., Cauthen, G.E., 1931. Viability of the eggs of the fowl nematode Ascaridia lineata (Schneider) exposed to natural climatic factors. J. Parasitol. 18, 113. Ackert, J.E., Cooper, R.M., Dewhirst, L.W., 1947. Viability of Ascaridia eggs under varying conditions of age and administration. Trans. Am. Microsc. Soc. 66, 383–389. Arene, F.O.I., 1986. Ascaris suum: influence of embryonation temperature on the viability of the infective larva. J. Therm. Biol. 11, 9–15, http://dx.doi.org/10.1016/0306-4565(86)90011-2. Bird, A.F., Bird, J., 1991. The egg. In: Structure of Nematodes, second ed. Academic Press, San Diego, CA, USA, pp. 7–43. Christenson, R.O., Earle Jr., H.H., Butler Jr., R.L., Creel, H.H., 1942. Studies on the eggs of Ascaridia galli and Heterakis gallinae. Trans. Am. Microsc. Soc. 61, 191–205. Denbow, D.M., 2000. Gastrointestinal anatomy and physiology. In: Whittow, G.C. (Ed.), Sturkie’s Avian Physiology. , fifth ed. Academic Press, San Diego, CA, USA, pp. 299–325.

107

Gaasenbeek, C.P., Borgsteede, F.H., 1998. Studies on the survival of Ascaris suum eggs under laboratory and simulated field conditions. Vet. Parasitol. 75, 227–234. Hansen, M.F., Oonyawongse, R., Ackert, J.E., 1953. Rate of development, viability, and virulence of Ascaridia galli ova cultured respectively in air and in water. Am. Midl. Nat. 49, 743–750. Höglund, J., Jansson, D.S., 2011. Infection dynamics of Ascaridia galli in non-caged laying hens. Vet. Parasitol. 180, 267–273. Jansson, D.S., Nyman, A., Vågsholm, I., Christensson, D., Göransson, M., Fossum, O., Höglund, J., 2010. Ascarid infections in laying hens kept in different housing systems. Avian. Pathol. 39, 525–532. Kaufmann, F., Das¸, G., Sohnrey, B., Gauly, M., 2011. Helminth infections in laying hens kept in organic free range systems in Germany. Livest. Sci. 141, 182–187. Lazdina, M.A., Grinberga, M.A., 1978. Effect of the pH of Ascaridia galli egg culture medium on the experimental infection in chickens. Angew. Parasitol. 19, 202–207. Levine, P.P., 1937. The viability of the ova of Ascaridia lineata when exposed to various environmental conditions. J. Parasitol. 23, 368–375. McRae, A., 1935. A study of the moisture requirements of the eggs of the chicken ascarid, Ascaridia galli. J. Parasitol. 21, 220. Nordin, A., Nyberg, K., Vinnerås, B., 2009. Inactivation of Ascaris eggs in source-separated urine and feces by ammonia at ambient temperatures. Appl. Environ. Microbiol. 75, 662–667. Permin, A., Bisgaard, M., Frandsen, F., Pearman, M., Kold, J., Nansen, P., 1999. Prevalence of gastrointestinal helminths in different poultry production systems. Br. Poult. Sci. 40, 439–443. Permin, A., Hansen, J.W., 1998. Epidemiology, Diagnosis and Disease Control of Poultry Parasites. FAO, Rome, Italy. Reid, W.M., 1960. Effects of temperature on the development of the eggs of Ascaridia galli. J. Parasitol. 46, 63–67. Roberts, F.S.H., 1937. Studies on the biology and control of the large roundworm of fowls, Ascaridia galli (Schrank 1788) Freeborn 1923. Qld. Agric. J. 47, 8–16. Saunders, L.M., Tompkins, D.M., Hudson, P.J., 2000. The role of oxygen availability in the embryonation of Heterakis gallinarum eggs. Int. J. Parasitol. 30, 1481–1485. Sherwin, C.M., Nasr, M.A.F., Gale, E., Petek, M., Stafford, K., Turp, M., Coles, G., 2013. Prevalence of nematode infection and faecal egg counts in free-range laying hens: relations to housing and husbandry. Br. Poult. Sci. 54, 12–23. Tongson, M.S., 1967. Development of viability of Ascaridia galli under different temperatures. Philipp. J. Vet. Med. 4, 56–61. Wharton, D.A., Allan, G.S., 1989. Cold tolerance mechanisms of the freeliving stages of Trichostrongylus colubriformis (Nematoda). J. Exp. Biol. 145, 353–369.