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.

This thesis describes a series of studies designed to investigate important aspects of chicken gastrointestinal nematodes with a focus on A. galli. The studies aimed broadly to i) evaluate methods for isolation, laboratory storage and in vivo maintenance and multiplication of stocks of A. galli for critical experimentation; ii) refine existing methods or develop new tools for anthelmintic efficacy testing in chicken nematodes; and iii) determine anthelmintic resistance profiles of Australian chicken nematode isolates on selected free-range egg farms.

The objective of the first study (Chapter 3) was to define the rate of egg production by mature female A. galli worms cultured in RPMI media, and evaluate changes in egg viability under different storage and incubation conditions. Eggs recovered after 1, 2 or 3 days of culture were subjected to either storage in water at 4°C (1, 4 or 8 weeks) followed by incubation in 0.1 N H₂SO₄ at 26°C (2, 4 or 6 weeks) or prolonged storage at 4°C for up to14 weeks. Of the 6,044 eggs recovered per mature female 49.2, 38.5 and 12.3% were recovered on days 1, 2 and 3 of worm incubation respectively with similar initial viability (≥99%) between days. It was observed that the storage and incubation conditions, not the day of egg recovery, are the main factors affecting A. galli egg viability. Prolonged storage at 4°C significantly reduced both the proportion of morphologically normal unembryonated eggs and their embryonation capacity resulting in decline in viability of 5.7-6.2% per week. A smaller but significant decline in egg (2.0%) and larval (1.4%) viability per week of incubation at 26°C was also observed.

The aim of the second study (Chapter 4) was to determine the most efficient means of multiplying stocks of A. galli eggs for further experimentation and maintenance of defined worm strains. A total of 384 layer cockerel chicks were used in an experiment with a 2 x 3 x 4 x 2 factorial design involving age of chicken at infection (day-old or 14 days old), immunosuppression (dexamethasone (DEX), cyclophosphamide (CY) or sham), infective egg dose (0, 100, 300 or 900 embryonated eggs/bird) and time of worm recovery after infection (8 or 10 weeks post-infection) was conducted. Body weight, excreta egg counts, intestinal worm count and worm establishment rate were assessed. Immunosuppression with DEX, but not CY resulted in significantly higher mean worm burden than in control chickens with excreta egg counts also considerably higher in DEX treated birds. Infection caused a significant dose dependent reduction in body weight in non-immunosuppressed birds but this effect was ameliorated by immunosuppression implicating the immune response in the reduced growth caused by infection. Age at infection had no significant effect on the studied variables although both worm and egg counts were numerically higher in the day-old infected groups. Egg dose significantly influenced the prevalence of infection, worm establishment rate, worm egg production and mean worm count. The 300 and 900 egg doses resulted in significantly higher worm count and egg production than the 100 egg dose. A significant negative correlation was observed between egg dose and worm establishment rate indicating an inverse relationship.

Chapters 5, 6 and 7 present the results of investigations into the efficacy of commercial anthelmintics against different nematode species and their developmental stages. Additionally, Chapter 7 assessed worm control practices by free-range egg farmers using an online survey comprising 36 questions implemented using SurveyMonkey. Controlled efficacy experiments were conducted using nematode isolates sourced from 5 different free-range farms with the objectives of i) evaluating the efficacy status of LEV, PIP, FBZ or FLBZ, and a levamisole-piperazine combination (LEV-PIP) against the worm isolates and their life cycle stages including in uteri ovicidal activity; ii) comparing the efficacy of individual oral administration against mass administration in drinking water (Chapters 5 and 6); and iii) comparing the EECR and WCR tests for determining anthelmintic efficacy in chickens under different infection modalities. Three infection models were employed in separate controlled trials. In Chapter 5 an A. galli isolate surviving a recent treatment with LEV (suspected case of LEV resistance) was used in an artificial infection model which produced adult warms only (mature worm infection model). In Chapter 6, two additional A. galli isolates originating from two different farms were tested using a split trickle infection model which produced mixed age A. galli populations at the time of treatment (adult, luminal larvae and histotrophic larvae). The viability of eggs isolated from adult A. galli worms expelled by treated birds was also assessed. The experiments in Chapter 7 employed layer chickens harbouring natural mixed infections of A. galli, H. gallinarum and capillaria spp sourced from two free-range farms. Chickens received label-recommended doses of LEV (28 mg/kg), PIP (100 mg/kg) or LEV-PIP co-administered at their full individual doses as a single oral drench or in group drinking water at recommended concentrations of 0.8 mg/ml or 2.5 mg/ml over eight hours for 1 and 2 days respectively, FLBZ (30 ppm or 60 ppm) in the feed over 7 days, and FBZ (Panacur 25® Sheep drench) at two dose rates (10 mg/kg as a single oral drench or 5mg/kg as 0.023 mg/ml in drinking water over eight hours for 5 days). Anthelmintic efficacies were estimated by both WCR% and EECR% 10 days after start of anthelmintic administration following World Association for the Advancement of Veterinary Parasitology guidelines. Based on a standard cut-off value of ≥90%, LEV, LEV-PIP, FLBZ and FBZ attained the desired efficacy against nematodes from all flocks but PIP exhibited a poor efficacy against immature A. galli (61-85%), all stages of H. gallinarum (42-77%) and capillaria spp (25-44%). A. galli worms expelled after treatment with LEV, PIP or their combination, but not FLBZ contained viable eggs. FLBZ provided 92 and 100% control of tapeworms at 30 and 60 ppm dose rates respectively in the single study with tapeworms. The online survey with a low response rate of 16/203, revealed that worm infection was of moderate concern to the producers and the majority (68%) felt that the current anthelmintics work effectively. Application of anthelmintics in water was generally effective with a numerically slightly lower efficacy than individual application. The EECRTs were inconsistent with the WCRT in infections involving mixed species and/or developmental stages.

Lastly the potential of in vitro drug sensitivity tests for use in A. galli were evaluated, aiming to i) optimise pre-assay sample preparation methods including extracting eggs from chicken excreta using different flotation fluids and comparing the deshelling centrifugation method and the glass-bead method with or without bile, to induce larval hatching in vitro and ii) evaluate two in vitro efficacy assays, the in-ovo larval development assay (LDA) and larval migration inhibition assay (LMIA) using fresh A. galli eggs and artificially hatched larvae, respectively. Four anthelmintics, namely thiabendazole (TBZ), FBZ, LEV and PIP were tested using a fully susceptible A. galli isolate sourced from a free-range farm. The results suggested that the LDA and LMIA could successfully be used to generate concentration-response curves for the tested drugs. The LDA was effective for the ovicidal benzimidazole anthelmintics with TBZ and FBZ having EC₅₀ values for inhibiting egg embryonation of 0.084 and 0.071 µg/ml, respectively. In the LMIA, the values of EC₅₀ of TBZ, FBZ, LEV and PIP were 105.9, 6.32, 349.9 and 6.78 × 10⁷ nM, respectively. For such in vitro studies, use of a saturated sugar solution to recover eggs resulted in high egg recovery efficiency (67.8%) and yielded eggs of the highest morphological quality (98.1%) and subsequent developmental ability (93.3%). The larval hatching methods evaluated did not differ in hatching efficiency but the deshelling-centrifugation method seemed to yield larvae with slightly better survival rates.

The results of this thesis demonstrated that A. galli eggs for experimentation can be effectively recovered from mature female worms or excreta collected from infected chickens where egg viability of 90% or above can be achieved in both cases. Post recovery storage and incubation conditions significantly affect A. galli egg viability. Trickle infection at day-old with infective doses of 300 eggs coupled with immunosuppression with DEX would provide the most efficient way to amplify and maintain A. galli worms in vivo, as using older birds or a higher egg dose did not provide any advantage. No evidence of loss of efficacy of the tested anthelmintics was detected on these farms reinforcing the perception of free range egg producers that participated in the online survey that current anthelmintics work effectively. The fact that eggs recovered from worms expelled after treatment with LEV, PIP or LEV-PIP retained embryonation capacity has epidemiological implications but may also provide an option for recovering eggs for A. galli propagation experiments. The EECR procedures are not suitable alternatives for evaluating anthelmintic efficacy in infections with mixed species or if efficacy against non-adult stages is required but could be useful to determine efficacy against adult nematodes in mono-species artificial infections. The preliminary studies into in vitro assays for testing anthelmintic efficacy against A. galli indicated significant potential in this area with the LDA and LMIA allowing for calculation of EC₅₀ values. However, standardization of these tests and establishment of EC₅₀ reference values and correlation with the WCR tests need to be performed although optimization of the methodologically complex LMIA may not be warranted for routine diagnosis of AR. Put together, the outcomes of this thesis can improve the ability of researchers, advisors and farmers to investigate and manage gastrointestinal nematode infections of chickens.