Helminth infections in laying chickens in Australia: prevalence, diagnosis, and improved methods of worm egg storage and multiplication

This thesis describes a series of studies designed to investigate important aspects of gastrointestinal helminth parasites in chicken. The studies aimed broadly to 1) provide compiled information about the status and trends of helminth infections in poultry operations worldwide and assess the prevalence and magnitude of helminth infections in commercial cage-free laying chickens in Australia" 2) Evaluate and optimise diagnostic tools for routine monitoring of nematode infections in chickens" 3) optimise prolonged laboratory storage methods for both undeveloped and embryonated stages of nematode eggs." and 4) evaluate the embryonation and infectious capacity of A. galli eggs isolated from excreta, worm uteri or worms cultured in artificial media. This thesis commences with Chapter 1 (General introduction) that contains an outline of the background, research problem, research aim, and propositions. The literature review chapter 2 provides an overview and summarises the key aspects of helminthiasis in chicken relevant to the research work and identify areas where knowledge is lacking. The objective of the first study (Chapter 3) was to provide an overview of the published information regarding the epidemiology and the diagnostic approaches of chicken helminth infection. Six databases were searched for studies and a total of 2,985 articles published between 1942 and 2019 were identified and subsequently screened for eligibility using title/abstract and full text assessment, resulting in 191 publications used in the study. Post-mortem diagnostics (73.8%) and the flotation technique (28.8%) were commonly used to detect helminth infections with pooled prevalence of 79.4%. More than 30 helminth species in chicken populations were identified including A. galli (35.9%), H. gallinarum (28.5%), Capillaria spp. (5.90%) and Raillietina spp. (19.0%) being the most prevalent. The reported prevalence of helminth infection decreased over time in developing countries while it increased in the developed world. Chicken kept in back yard and free-range systems had a markedly higher pooled prevalence of helminth infection (82.6 and 84.8%, respectively) compared to those housed in cage production systems (63.6%). The aim of the second study (Chapter 4) was to determine the prevalence and worm burdens of intestinal helminth infection in cage-free laying chickens in Australia. In an online survey of worm prevalence, a high proportion of respondents reported detection of Ascaridia galli (77%), followed by tapeworms (69%) and caecal worms (Heterakis gallinarum) (62%), whereas fewer respondents (23%) reported the presence of hair worms (Capillaria spp.) in their flocks. Total worm recovery from 407 laying hens on four farms found that 92.1% of hens harboured one or more helminth parasite with a prevalence of 73 to 100% across farms. Mixed infections were common with 79% of hens harbouring two or more helminth species. The prevalence of nematode species H. gallinarum, A. galli and Capillaria spp. was 87, 82 and 35% respectively, whereas the overall prevalence of the cestodes was 12%. The hens harboured an average of 71 worms with H. gallinarum having the highest mean burden (45.5 worms/hen) followed by A. galli (22.0 worms/hen), Capillaria spp. (2.7 worms/hen) and cestodes (0.8 worms/hen). When investigating intestinal excreta (n = 10) and caecal excreta (n = 10) of 16 flocks, all sampled flocks were egg count positive for ascarid infections, predominantly A. galli and H. gallinarum, respectively. The aim of the third study (Chapter 5) was to assess and optimise laboratory and field sampling methods for routine monitoring of nematode infections in chickens by evaluating the sensitivity, accuracy, and precision of the Modified McMaster (MM) and Mini-FLOTAC (MF) methods using laying chicken excreta samples spiked with estimated true numbers of eggs (Experiment 1 = 5-1500 EPG (eggs/g)" Experiment 2 = 5-500 EPG) without and with operator effects, respectively or using individual fresh excreta (n = 230) and fresh floor excreta (n = 42) from naturally infected free-range layer farms. The Coefficient of Variation was assessed within and between operators and the time spent on sample preparation and counting was also evaluated. MM was more accurate than MF, particularly at higher EPG levels, but slightly less precise and sensitive, particularly at low EPG levels, while taking less laboratory time per sample. Our observations indicate that the MM method is more appropriate for rapid diagnosis of chicken nematodes in the field. Pooled fresh floor excreta samples would be sufficient to indicate infection level in free range farms. The aim of the fourth study (Chapter 6) was to determine ideal storage conditions for maximising the viability of A. galli eggs and maintaining viability for the longest period. A 2 x 2 x 3 x 5 factorial experimental design was employed to investigate the effects of storage temperature (4˚C or 26˚C), storage condition (aerobic or anaerobic), storage medium (water, 0.1 N H2SO4 or 2% formalin) and storage period (4, 8, 12, 16 and 20 weeks). The viability of eggs was assessed after eggs in all treatments were held aerobically at 26˚C for 2 weeks after the storage period to test embryonation capacity. The maintenance of viability during storage at 4˚C was optimal under anaerobic conditions while at 26˚C it was optimal under aerobic conditions. Anaerobic conditions at 26˚C led to a rapid loss of viability while aerobic conditions at 4˚C had a less severe negative effect on maintenance of viability. Egg storage in 0.1 N H2SO4 resulted in a significantly higher viability overall (54.7%) than storage in 2% formalin (49.2%) or water (37.3%). Untreated water was the least favourable storage medium when eggs were stored at 26˚C while it was a medium of intermediate quality at 4˚C. The lowest rate of decline was seen with storage of eggs under anaerobic conditions at 4˚C or aerobic conditions at 26˚C in 0.1 N H2SO4 with a decline rate of approximately 2% per week with no significant difference between the two. Therefore, this study has clearly revealed anaerobic conditions required for prolonged storage of A. galli eggs in the pre-embryonated state at 4˚C. It has also identified that 0.1 N H2SO4 provides the best preservation against degradation during storage, particularly at 26˚C under aerobic conditions. The aim of the final study (Chapter 7) was to compare the infectivity of A. galli eggs isolated from A. galli egg sources (worm uteri, excreta or eggs shed in vitro) under two infection regimens. A 3x2 factorial arrangement was employed to test the infectivity of A. galli eggs from the three sources and two modes of infection (single or trickle infection). One hundred and fifty-six Isa-Brown one day-old cockerels randomly assigned to the six treatment groups (n = 26) were orally infected with embryonated A. galli eggs obtained from the three A. galli egg sources (worm uteri, excreta or eggs shed in vitro) administered either as single dose of 300 eggs at one day-old or trickle infected with 3 doses of 100 eggs over the first week of life. Eggs obtained from cultured worms or excreta exhibited a higher embryonation capacity than eggs obtained from worm uteri. The findings showed that eggs shed by cultured worms or isolated from worm uteri had greater infective capacity than eggs harvested from excreta and that trickle rather than single infection resulted in higher worm establishment rate