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Introduction Poultry production in Denmark and other industrialised countries has gone through major changes since the 1950'es due to the development of new technology, abolition of tariff barriers and international competition. From mainly being a “backyard business”, poultry is now being produced intensively at relatively few, but large units consisting of up to 275,000 birds. Today, Denmark has a production of approximately 80 million kilogram eggs and 130 million broilers per year (Anon., 2002). In Denmark, recent changes in consumer demands have resulted in an increasing number of table eggs being produced in free-range systems. The production of broilers in free-range systems are also increasing, although not yet fully established due to bacterial as well as parasitic problems. Production losses are significantly higher in free-range production systems compared to conventional intensive indoor production systems. No systematic analysis of the cause of the overall mortality and morbidity in free- range production systems is available at present. However, a crosssectional study has shown that infections with intestinal roundworms such as A. galli, H. gallinarum and C. obsignata are highly prevalent in free-range systems (Permin et al. , 1999). Infections with such endoparasites have been estimated to cause production losses in the range of 10-20 % due to impaired feed conversion, reduced growth and egg production, and increased mortality (Ikeme, 1971; Soulsby, 1982). H. gallinarum is of additional importance due to its ability to transfer the protozoa Histomonas meleagridis , which causes Blackhead in poultry (Soulsby, 1982). Furthermore, a study have shown that Salmonella enterica can be transferred by eggs from A. galli to chickens (Chadfield et al. , 2001). Also, ongoing investigations suggest that A. galli have a negative impact on concurrent infections with Pasteurella multocida and E. coli (Dahl et al ., 2002; Permin et al ., 2002). The high prevalence of parasites in free-range systems is probably a result of several factors. The animals have access to outdoor areas where a range of parasites have optimal conditions, and where the increased contact to wildlife constitute an additional risk of contamination of the farm. Also, the typical large number of animals in the flocks, together with regulations in force that have prohibited the use of prophylactic anthelmintics, greatly enhances the risk of parasitic diseases. Therefore, there is a marked need for alternative methods of parasite control in free-range poultry production systems. The dry powder Stalosan F is used in pig, cattle and poultry houses as a disinfectant. Several studies have demonstrated the capability of Stalosan F to reduce numbers of bacteria, virus and fungi (Anon., 2000). Furthermore, it has been shown that the disinfectant has an effect on sporulation rates of coccidian oocysts (Anon., 2000). These findings have raised expectations that Stalosan F might have an effect on eggs from other parasites, and that the disinfectant consequently can function as an alternative or a supplement to conventional control of parasites in free-range poultry production systems. The main purpose of the present study was therefore to evaluate the effect of Stalosan F on the commonly occurring poultry parasites A. galli, H. gallinarum , and Capillaria spp. in three experiments. First, the effect of in vitro application of Stalosan F to different developmental stages of the parasite eggs was evaluated. Secondly, it was examined whether this treatment had any effect on the infectivity of the eggs. The third experiment assessed the impact of applying Stalosan F to a parasite egg contaminated environment on infection of chickens. Materials and Methods Parasite material and isolation of mixed parasite eggs from faeces Twenty hens, excreting A. galli, H. gallinarum and Capillaria spp. eggs, were purchased from an organic egg production system and caged. Approximately 5 kilogram fresh faeces was collected from the hens during a 48 hour period. The pooled faeces was mixed with tap water before being washed through a series of sieves with mesh apertures of 200, 90, 20, respectively. The material (including parasite eggs) retained in the last sieve was collected and kept in a small volume of water. After counting the eggs, the suspension was diluted to contain 2500 eggs/ ml and then divided into three portions: One portion was stored at 5°C to prevent embryonation of the eggs (two days later, this portion was used to evaluate the effect of Stalosan F on newly excreted parasite eggs). The second portion was stored in 0.1 N sulphuric acid (H 2 SO 4 ) according to the method described by Permin et al . (1997 a ) at 18°C for 5 weeks to embryonate in order to study the effect of the disinfectant on the infective L 3 -stage of the parasites. The third portion was also stored at 5°C to prevent embryonation of the eggs. These eggs were later used in an experimental infection to evaluate the effect of Stalosan F on the infectivity of parasite eggs.
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Isolation of A. galli eggs from adult female worms A batch of A. galli eggs was obtained according to a method described by Permin et al. (1997 a ). In short, the hens were slaughtered and adult female A. galli collected from the small intestine. The worms were dissected and their uteri transferred to a Petri dish where the eggs were squeezed out using a wooden spatula. Tissue debris were removed and the eggs mixed with a small amount of tap water. The eggs were counted and the suspension diluted to contain 2500 eggs/ml and then divided into two portions. One portion was stored at 5°C to prevent embryonation of the eggs. Two days later, this portion was used to evaluate the effect of Stalosan F on nonembryonated A. galli eggs. The second portion was stored in 0.1 N sulphuric acid at 18°C for 5 weeks until the eggs were embryonated in order to study the effect of the disinfectant on the infective L 3 -stage of A. galli . Experiment 1: in vitro treatment and examination The effect of in vitro treatment with Stalosan F was examined and compared with commercial bird sand as control for all four batches of parasite eggs: 1) newly excreted non-embryonated mixed eggs; 2) embryonated mixed eggs; 3) freshly extracted nonembryonated A. galli eggs; and 4) embryonated A. galli eggs. A 300 l egg suspension (~750 eggs) was spread over a layer of neutral agar in labelled Petri dishes. Stalosan F or bird sand was then sprinkled evenly over the egg suspension in the Petri dishes at a concentration of 100 g/m 2 and incubated at 18°C for 1, 2, 3, 7 or 21 days (see Table 1). After the required exposure time the eggs were washed off the Petri dishes in tap water. The disinfectant or sand was subsequently removed by a light spin in the centrifuge leaving the eggs in the supernatant. The eggs were examined under microscope and judged either as “normal appearing” or “abnormal”. The experiment was done in triplicate. Experiment 2: Experimental infections A batch of parasite eggs was made to evaluate the effect of Stalosan F on the infectivity of newly excreted mixed parasite eggs. Half of the batch was, together with the disinfectant (100 g/m 2 ), applied over a layer of neutral agar in large Petri dishes and incubated at 18°C for one week, after which the parasite eggs were washed in tap water and then incubated in 0.1N sulphuric acid at 18°C. The other half of the batch was treated similarly, except that commercial bird sand was used instead of the disinfectant. After 4 weeks of further incubation (i.e. a total of 5 weeks), the eggs were counted and diluted to contain 1000 eggs/ml in both batches and two groups of 25 parasitenaive chickens, 4 weeks of age, were subsequently inoculated with 500 eggs. Group 1 received the Stalosan F -treated eggs and group 2 received the untreated eggs. All animals were slaughtered 8 weeks after inoculation, and their intestines examined for the presence of parasites (Permin and Hansen, 1998).
