INTRODUCTION
Ascaridia galli (Schrank, 1788), which is a common parasitic nematode of chickens, has a worldwide distribution (Kaufmann, Reference Kaufmann1996). This parasite has a direct life cycle and chickens become infected by ingestion of infective eggs containing the infective larval stage which has been referred to as either second-stage larvae (L2) (Moran and Mizelle, Reference Moran and Mizelle1957; Herd and McNaught, Reference Herd and McNaught1975; Ramadan and Abou Znada, Reference Ramadan and Abou Zanda1992) or third-stage larvae (L3) (Ackert, Reference Ackert1931; Araujo and Bressan, Reference Araujo and Bressan1977). After ingestion, the eggs hatch in the duodenum and the infective larvae are released in the intestine (Ackert, Reference Ackert1923). Most of the larvae then undergo a so-called histotropic or mucosal phase, starting as early as 1 day post-infection (d.p.i.) (Tugwell and Ackert, Reference Tugwell and Ackert1952). The duration of this phase has been reported to vary from 2 to 7 weeks depending on the level of infection (Herd and McNaught, Reference Herd and McNaught1975) but whether some larvae stay in this phase for an extended period of time is largely unknown. The adult parasites reside in the lumen of the intestine (Ackert, Reference Ackert1923, Reference Ackert1931; Tugwell and Ackert, Reference Tugwell and Ackert1952). Generally the pre-patency period is reported as 5–8 weeks (Ackert, Reference Ackert1931; Kerr, Reference Kerr1955). Infection at any age is associated with substantial economic losses due to a decrease in growth rate and weight loss. Moreover, the larvae may damage the intestinal mucosa and a heavy infection with adult worms can obstruct the small intestine and cause death (Ackert and Herrick, Reference Ackert and Herrick1928; Ramadan and Abou Znada, Reference Ramadan and Abou Zanda1991; Phiri et al. Reference Phiri, Phiri, Ziela, Chota, Masuku and Monrad2007). Ascaridia galli may also play a role in transmission of other infections such as Salmonella in chickens (Chadfield et al. Reference Chadfield, Permin, Nansen and Bisgaard2001) and not least cause aesthetic problems when worms are recovered in eggs by consumers, creating problems for the industry (Fioretti et al. Reference Fioretti, Veronesi, Diaferia, Franciosini and Proietti2005).
Recent European regulation for the protection and welfare of laying hens substituted the use of traditional (conventional unenriched cage) systems with enriched cage or floor husbandry systems with or without access to outdoor runs. Moreover, the attitudes of consumers towards animal welfare and organic food products have also increased the production in organic farming systems (1999/74/EC, Anonymous, 1999). These floor-housing systems together with the direct life cycle and the highly resistant nature of the eggs all favour the transmission of this parasite (Permin and Hansen, Reference Permin and Hansen1998; Permin et al. Reference Permin, Bisgaard, Frandsen, Pearman, Kold and Nansen1999; Kaufmann et al. Reference Kaufmann, Das, Sohnrey and Gauly2011). Permin et al. (Reference Permin, Bisgaard, Frandsen, Pearman, Kold and Nansen1999) reported that in Denmark A. galli prevalence in the free-range system was higher (63·8%) than in the deep-litter system (41·9%), the backyard system (37·5%) and the conventional cage system (5%). In Sweden the prevalence of A. galli in different housing systems in 2008 was 77·1% in free range and organic systems, 28·6–52·2% in different kinds of floor systems and 4·3% in cage systems (Jansson et al. Reference Jansson, Vågsholm, Nyman, Christensson, Göransson, Fossum and Höglund2010). Similarly, in Germany, a high prevalence (88%) of A. galli was reported in organic free-range systems (Kaufmann et al. Reference Kaufmann, Das, Sohnrey and Gauly2011) and in Ethiopia, Abebe et al. (Reference Abebe, Asfaw, Genete, Kassa and Dorchies1997) found a higher prevalence of A. galli (71·6%) in free-range chickens followed by 49% in semi-intensive and 0% in cage systems.
Most of these studies carried out on the population biology of A. galli infection include only single-time necropsy at the time of patency and therefore do not provide information about the dynamics of the establishment of the infections (Permin et al. Reference Permin, Bojsen, Nansen, Bisgaard, Frandsen and Pearman1997; Gauly et al. Reference Gauly, Homann and Erhardt2005; Höglund and Jansson, Reference Höglund and Jansson2011). Thus more information on the establishment of A. galli before patency period is needed in order to understand the population dynamic of the parasite and the host response to infection in the early phase of infection. Such information is crucial in order to implement effective control measures and to improve the overall performance and productivity of the bird. Therefore, this study was carried out to study the population dynamics of A. galli following single infection by serial necropsy of birds from 3 to 42 days post-infection.
MATERIALS AND METHODS
Experimental animals
Seventy ISA Brown chickens obtained from a commercial breeding and pullet-raising farm with no history of A. galli infection were used in this experiment. The chickens were 6 weeks of age and weighed 0·48 kg±0·06 kg (mean±s.d.) at the start of the experiment. The chickens were randomly allocated into two groups consisting of 28 chickens in the control group and 42 in the infected group. They were housed in three pens separated from each other by at least 2 metres. All the control birds were placed in one pen and the birds of the infected group were housed in the other two pens. The birds were offered commercial pullet feed 3 times per day and had access to water and grit ad libitum.
Collection and preparation of the infection dose
Approximately 1 kg of fresh chicken feces was collected from a conventional indoor deep-litter layer farm with a known high prevalence of A. galli and the eggs were isolated by wet sieving according to Ferdushy et al. (Reference Ferdushy, Nejsum, Roepstorff, Thamsborg and Kyvsgaard2012). The collected eggs were then set for embryonation in 0·05 m H2SO4 (pH 1 ) in a culture flask at 22 °C kept in the dark for 6 weeks. Once a week, the eggs were aerated for 15 min and the rate of embryonation was checked. After embryonation the eggs were incubated at 5 °C until use (not more than 2 weeks after embryonation).
Experimental design
After a week of adaptation, chickens in both groups were allocated randomly into 7 necropsy dates (3, 7, 10, 14, 21, 28 and 42 d.p.i.) after stratification for body weight. Therefore, for each time-point there were 10 birds (4 controls and 6 infected). Birds from each group were marked by use of different coloured leg bands and individual wingtag numbers. On day 0, all the birds forming the infected group were inoculated with 500 embryonated A. galli eggs suspended in 1 mL of tap water using a plastic Pasteur pipette inserted via the oesophagus to the level of the crop. The vial with the inoculation dose was washed twice with 1 mL of water that was given to the birds by the same pipette, as per Ferdushy et al. (Reference Ferdushy, Nejsum, Roepstorff, Thamsborg and Kyvsgaard2012). The birds were not given the morning feed until after the infection had taken place. Body weight and fecal samples were taken from all birds of both control and infected groups 1 day before the inoculation and again at necropsy. Collected fecal samples were examined for the presence of helminth eggs by a modified McMaster method with a lower detection limit of 20 eggs per gram (EPG) of feces (Roepstorff and Nansen, Reference Roepstorff and Nansen1998).
Necropsy and larval recovery from the intestinal content and wall
At each time-point (3, 7, 10, 14, 21, 28, 42 d.p.i.) respective numbers of infected and control chickens were killed by decapitation. The gastrointestinal tract was removed from the proventriculus to the cloaca, divided into two main sections; (i) duodenum (defined by the duodenal loop and referred to as section D) (Schummer et al. Reference Schummer, Vollmerhaus, Sinowatz, Frewein, Waibl, Nickel, Schummer and Seiferle1992) and (ii) jejuno-ileum (from entry of the bile duct to the origin of caeca as defined by Schummer et al. Reference Schummer, Vollmerhaus, Sinowatz, Frewein, Waibl, Nickel, Schummer and Seiferle1992) and divided into four equally sized subsections (J1, J2, J3, J4). Each intestinal section was opened separately in the longitudinal direction and washed by dipping the intestinal wall 10 times in 150 mL of 0·9% NaCl solution (38 °C). The washing water together with the intestinal contents was embedded in agar and incubated as described by Ferdushy et al. (Reference Ferdushy, Nejsum, Roepstorff, Thamsborg and Kyvsgaard2012). In short the samples were mixed with 150 mL of 2% agar solution (equivalent to 1% agar in the final solution), and immediately poured onto a humid agar cloth (45104S, Johnson's Universalduk, Johnson and Johnson AB, Sweden) placed on a tray and allowed to solidify for a few minutes at room temperature. Approximately 15 min elapsed between slaughter of the bird and setting the sample in agar-gel. The agar gels were incubated in warm physiological saline overnight at 38 °C. The following day larvae (no adult worms were found) were collected on a 15 μm sieve and stored in 70% alcohol until counting.
The rinsed sections of intestinal wall were further processed by artificial pepsin-HCl digestion (12 mL HCl (30%), 30 mL liquid pepsin (660 U per mL, Orthana Biofac A/S, Denmark) in 1 L of 42 °C tap water) as per Ferdushy et al. (Reference Ferdushy, Nejsum, Roepstorff, Thamsborg and Kyvsgaard2012). Briefly, the small intestinal wall was cut into small pieces of 0·5 cm and digested in 200 mL of digestion fluid under constant magnetic stirring of 250 rpm at 38 °C for 90 min or until full digestion of the tissue. Then the larvae were collected on a 15 μm sieve and stored in 70% alcohol until counting.
Data analysis and statistical methods
The statistical analyses were performed using SAS 9.1 and GraphPad Prism (version 5). Graphical presentations were made using GraphPad Prism (version 5) and Microsoft Excel 2007. The total number of larvae recovered at different d.p.i. were analysed in a Generalized Linear Model (PROC GENMOD procedure) specifying the negative binomial distribution in larval recovery and considering source (content and wall), section (D, J1, J2, J3, J4) and d.p.i. (3, 7, 10, 14, 21, 28, 42) as explanatory variables. Moreover, the interactions between d.p.i. and source, and d.p.i. and section, were also evaluated. The level of significance was considered as P < 0·05. Larval recovery over time was modelled by a simple exponential function with a constant rate of decay:
$$Y_t = Y_0 \times e^{ - K \times t} $$where Y t is the number of larvae recovered at time t, Y 0 is the estimate of the number of larvae established at t = 0, e is the exponential function with the rate of decay K.
RESULTS
Performances and prevalence
During the experimental period no clinical sign of infection was observed among the birds and also the fecal egg counts were negative for all the infected and control birds. No significant differences between groups were found with regard to weight gain (P=0·6). The prevalence of infection was 100% at all d.p.i. and the mean (±s.d.) number of larvae recovered were (100±17·4), (78±22·6), (90±10·2), (75±34·5), (55±13·8), (61±27·2) and (53±20·8) at 3, 7, 10, 14, 21, 28 and 42 d.p.i., respectively (Fig. 1). The recovery of larvae over time was modelled by an exponential function with a constant rate of decline (K=0·01832 with 95% CI 0·009221 to 0·02742) (Fig. 1). All the control birds were found to be uninfected.
Total number of larvae recovered at different days post-infection ( d.p.i.) from chickens infected with 500 embryonated Ascaridia galli eggs. The triangles represent larval counts from individual chickens, where the curve represents an exponential function with a constant rate of decay and a half-life of 37·8 days. Some data points were overlapping. To present these points they have been separated manually. The curve was estimated from the original data.
Distribution of larvae between the intestinal content and wall
The mean percentage recovery of larvae between the intestinal content and intestinal wall after digestion differed significantly among the d.p.i. (P < 0·0001). Almost all (70–86%) worms were obtained from the intestinal wall in the youngest infection (3–14 d.p.i.), but this fraction was reduced at 21 and 28 d.p.i. (62 and 51% respectively) (Fig. 2). At the last sampling date at 42 d.p.i. there was again a higher proportion in the wall. Comparatively larger larvae were obtained from the intestinal content than from the intestinal wall. The mean approximate length of the larvae in the content increased until 28 d.p.i. whereafter a decrease was observed at 42 d.p.i. but the mean lengths of the larvae in the wall were almost similar throughout the experimental period (Fig. 2).
Larval distribution between intestinal content and wall, and approximate length at different days post-infection (d.p.i.) of chickens infected with 500 embryonated Ascaridia galli eggs. Right y-axis: mean length (mm) of larvae obtained from the intestinal content and wall. Left y-axis: proportion of larvae in each of the two locations.
Distribution and size of larvae in different sections of the intestine
The number of larvae recovered from the intestinal sections differed significantly among the d.p.i. (P < 0·0001). Most of the larvae were located in section J2 at 3 and 7 d.p.i. From 10 d.p.i. onwards the larvae were located posteriorly being most abundant in section J3, and at 42 d.p.i. the highest number of larvae was again seen in section J2 (Fig. 3).
Total number of larvae recovered from different sections of the intestinal wall at different days post-infection ( d.p.i.) of chickens infected with 500 embryonated scaridia galli eggs. For definitions of sections D and J1–J4 see the Materials and Methods section. Six chickens were necropsied at each time-point.
The size of the larvae also varied greatly according to the d.p.i. (P < 0·0001). All the larvae recovered at 3 d.p.i. were < 1 mm. A large proportion of the larvae were growing. However, small larvae of a length below 1 mm were present at all necropsies (Fig. 4) and a mixture of different sizes of larvae was observed in different sections of the intestine at all d.p.i. except 3 d.p.i.
Total larval recovery according to size (mm) from 6 chickens infected with 500 embryonated Ascaridia galli eggs and necropsied at different days post-infection (d.p.i.).
DISCUSSION
This study investigated the development and localization of A. galli larvae during the pre-patent period. Within the 6-week study period no worms reached a size close to the mature fertile stage (51–76 and 72–116 mm for male and female, respectively) (Ackert, Reference Ackert1931). Differential development of the larvae was observed from 7 d.p.i. onwards, and from 21 d.p.i. a bimodal size distribution was evident. Although the proportion of larvae found in the intestinal contents was increasing with time (except for the last necropsy), more larvae were consistently recovered from the intestinal wall after pepsin digestion than from the contents. The mean lengths of larvae recovered from the intestinal contents were larger than those of the intestinal wall, and the larvae isolated from the wall did not seem to grow in size with time but remained more or less 1 mm. Other authors have also found this kind of small-sized larvae at late stages of infection (around 7 weeks) and referred to them as ‘static’ or ‘arrested larvae’ (Moran and Mizelle, Reference Moran and Mizelle1957; Herd and McNaught, Reference Herd and McNaught1975). According to Michel (Reference Michel1974) a bimodal size distribution after a single infection is indicative of arrested development and it is suggested to be related to three factors (or combinations hereof): (1) pre-determination induced by prior environmental exposure; (2) crowding effect/density dependency or (3) acquired immunity. Pre-determined arrested or inhibited development is a well-known phenomenon for the trichostrongylid nematodes in ruminants and is presumably induced in pre-parasitic L3 after exposure to seasonal changes in the environment (e.g. shorter photoperiod, decreasing temperature or humidity) (Eysker, Reference Eysker1997). Our study was carried out during the summer time in Denmark (June–July) and the lighting schedule followed the normal daylight. Thus, in this experiment it is difficult to explain the background for arrested development because of the environmental influence, but it cannot be ruled out that storage under refrigeration had induced this arrest, similar to trichostrongyles (Eysker, Reference Eysker1981). However, we have not experienced this after prolonged storage of other ascarids, e.g. Ascaris suum, and this remains hypothetical.
A reduced size of worms and resting stages due to crowding effect/density dependency for A. galli has been observed by Herd and McNaught (Reference Herd and McNaught1975). They infected two groups of chickens either with 2000 eggs or with 50 eggs and in the high-dose group arrested larvae were found until 54 d.p.i. whereas in the low-dose group arrested larvae were observed only until 16 d.p.i. Although we used a lower inoculation dose compared with the higher dose (500 vs 2000) given by Herd and McNaught (Reference Herd and McNaught1975) it can be speculated that the actual numbers of infective eggs in the two experiments were close as we used matured eggs collected from droppings which may have had a higher infectivity. This density-dependent development was also evident for other parasites e.g. Teladorsagia circumcincta in sheep (Hong et al. Reference Hong, Michel and Lancaster1986) and Oesophagostomum dentatum in pigs (Christensen et al. Reference Christensen, Barnes, Nansen, Roepstorff and Slotved1995).
The presence of smaller-sized larvae on all occasions may well be explained by acquisition of immunity. Comparable findings were also obtained by Idi et al. (Reference Idi, Permin and Murrell2004) who infected 4-week-old layer chickens with 500 embryonated A. galli eggs and only larval stages were recovered up to 10 weeks post infection. Similarly, Gauly et al. (Reference Gauly, Homann and Erhardt2005) infected four groups of chickens with A. galli eggs at the age of 6 weeks, 12 weeks, 18 weeks and 24 weeks and noticeably shorter and lighter worms were obtained from the 6-week-old group. Our chickens were 7 weeks of age at the time of infection and the findings of Idi et al. (Reference Idi, Permin and Murrell2004) and Gauly et al. (Reference Gauly, Homann and Erhardt2005) indicate that the birds were immunocompetent at that age. Herd and McNaught (Reference Herd and McNaught1975) also documented the effect of host immunity on A. galli growth and development as the proportion of arrested larvae was considerably lower in the birds treated with an immunosuppressive agent.
It remains speculative how the two subpopulations interact; the arrested population associated with the mucosa and the luminal population with larger and growing worms. The latter may be recruited from the first, but we do not know. We found no adult nematodes and can only assume that the luminal population will develop into adults with time as observed for Ostertagia ostertagi in both naturally and experimentally infected cattle (Michel et al. Reference Michel, Lancaster and Hong1976a, Reference Michel, Lancaster and Hong1976b).
We found that larvae were displaced more aborally with time. During the first week of infection most of the larvae were located in section J2 (i.e. the second quarter of the jejunoileum) similar to the findings of Ackert (Reference Ackert1923, Reference Ackert1931), Herd and McNaught (Reference Herd and McNaught1975) and Ferdushy et al. (Reference Ferdushy, Nejsum, Roepstorff, Thamsborg and Kyvsgaard2012). From 10 to 28 d.p.i. the larvae were located more posteriorly mainly in section J3 but at 42 d.p.i. they were again primarily found in section J2. Moran and Mizelle (Reference Moran and Mizelle1957) also obtained the majority (∼64%) of the recovered larvae from the section corresponding to section J3 in our study from 9 to 25 d.p.i. This caudal translocation of larvae from 10–28 d.p.i. in our study might be related to expulsion of larvae. Similarly, Roepstorff et al. (Reference Roepstorff, Eriksen, Slotved and Nansen1997) reported that in A. suum-infected pigs, larvae moved more caudally during the initial expulsion phase (14 to 21 d.p.i.) while they were again found in the oral part of small intestine from 28 d.p.i. and onwards.
The number of larvae was found to decline with time at a constant rate. The decrease in larval recovery over time may be influenced by the development of immunity and losses from other reasons e.g. expulsion or death of the larvae. Ascaridia galli-infected birds can develop specific IgG antibody and express a Th2 type response against A. galli antigens 14 days after infection (Degen et al. Reference Degen, van Daal, Rothwell, Kaiser and Schijns2005; Marcos-Atxutegi et al. Reference Marcos-Atxutegi, Gandolfi, Arangüena, Sepúlveda, Arévalo and Simón2009). The innate immunity of the bird may be responsible for the relatively low initial establishment (20% of the infection dose) and the later development of acquired immunity may play a role in the gradual elimination of the worms during the pre-patent period.
Our results may differ from the previous findings of other researchers as the method we employed was much more sensitive for the recovery of small larvae from the chicken intestine (Ferdushy et al. Reference Ferdushy, Nejsum, Roepstorff, Thamsborg and Kyvsgaard2012) and also because of the dissimilarities in other factors such as infection dose, breed, age and nutrition of the host (Permin et al. Reference Permin, Bojsen, Nansen, Bisgaard, Frandsen and Pearman1997; Schou et al. Reference Schou, Permin, Roepstorff, Sørensen and Kjær2003; Gauly et al. Reference Gauly, Homann and Erhardt2005; Idi et al. Reference Idi, Permin, Jensen and Murrell2007). During the experimental period no clinical signs were observed in control or infected birds, and both groups were gaining weight equally over time. This could be related to the absence of patent infection and to good management.
In conclusion it can be seen that the initial establishment of A. galli infection is much lower compared with other nematodes of monogastrics e.g. A. suum and Trichuris suis (Roepstorff et al. Reference Roepstorff, Eriksen, Slotved and Nansen1997; Kringel and Roepstorff, Reference Kringel and Roepstorff2006) where more than 50% of the inoculation dose can be recovered as larvae within 2–3 weeks. Of the established A. galli larvae, half seem to be expelled during 3–42 d.p.i. These two observations might be related to host immunity. We observed a lack of patency but the presence of two subpopulations of A. galli in the chickens – a small luminal population of increasing length (size) and a larger population of inhibited larvae attached to the gut wall, only recoverable after digestion. How these two subpopulations interact will require detailed long-term studies in order to understand the parasite population dynamics and host–parasite relationship more clearly.
ACKNOWLEDGEMENTS
The skilful help of L. Christiansen is greatly appreciated. All the birds were treated according to Danish legislation (License: A. Roepstorff, 2010/561-1914, 13 October 2000).
