Soil moisture influences the development of Haemonchus contortus and Trichostrongylus colubriformis to third stage larvae

Veterinary Parasitology 196 (2013) 161–171

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Soil moisture influences the development of Haemonchus contortus and Trichostrongylus colubriformis to third stage larvae S. Khadijah a,b,∗ , L.P. Kahn a , S.W. Walkden-Brown a , J.N. Bailey a , S.F. Bowers a a b

Animal Science, School of Environmental and Rural Science, University of New England, Armidale, NSW 2351, Australia Department of Biological Sciences, Faculty of Science and Technology, Universiti Malaysia Terengganu, 21030 Kuala Terengganu, Terengganu, Malaysia

a r t i c l e

i n f o

Article history: Received 14 September 2012 Received in revised form 13 January 2013 Accepted 15 January 2013 Keywords: Moisture Haemonchus contortus Trichostrongylus colubriformis Third stage larvae Faecal moisture Soil moisture Simulated rainfall

a b s t r a c t Two climate chamber experiments were conducted to determine the effect of varying initial soil moisture (0, 10 and 15%), simulated rainfall amount (0, 12 and 24 mm) and simulated rainfall timing (days −1, 0 and 3 relative to faecal deposition) on development (day 14) of Haemonchus contortus and Trichostrongylus colubriformis to the third stage larvae (L3) and faecal moisture (FM). Increasing initial soil moisture content from 0 to 10 or 15% led to higher recovery of total L3 (P < 0.001). Total L3 recovery increased with each level of simulated rainfall (P < 0.001) in the ascending order of 0, 12 and 24 mm. There was an interaction between the effects of initial soil moisture and simulated rainfall amount on the recovery of total L3, showing that the benefit of increased simulated rainfall lessened with increasing soil moisture. Simulated rainfall on the day of deposition resulted in higher recovery of L3 (P < 0.001) than simulated rainfall on other days. FM on day 3 relative to faecal deposition was best associated with recovery of total H. contortus and T. colubriformis L3 (R2 = 0.32–0.46), reinforcing the importance of sufficient moisture soon after faecal deposition. The effects of initial soil moisture, and the amount and timing of simulated rainfall on development to L3 were largely explained by changes to FM and soil moisture values within 4 days relative to faecal deposition. These results highlight the influence of soil moisture and its interaction with rainfall on development of H. contortus and T. colubriformis to L3. Consequently we recommend that soil moisture be given greater importance and definition in the conduct of ecological studies of parasitic nematodes, in order to improve predictions of development to L3. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Haemonchus contortus and Trichostrongylus colubriformis are the two most important parasitic gastrointestinal nematodes (GIN) of sheep in tropical and temperate summer rainfall areas. The development of the free-living stages of these nematodes depends on environmental

∗ Corresponding author at: Animal Science, School of Environmental and Rural Science, University of New England, Armidale, NSW 2351, Australia. Tel.: +61 0267732586; fax: +61 0267733922. E-mail addresses: [email protected], [email protected] (S. Khadijah). 0304-4017/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetpar.2013.01.010

variables, mainly temperature and moisture. When the temperature is suitable for development, sufficient moisture is needed to ensure successful development to the third stage larvae (L3). Moisture available in the faeces is more important for the development of eggs to L3 for H. contortus than T. colubriformis as the egg shell of H. contortus is more permeable to water at all temperatures (Waller, 1971), making this species more suceptible to dessication (O’Connor et al., 2006). Rainfall timing relative to faecal deposition (Barnes et al., 1988; Besier and Dunsmore, 1993; O’Connor et al., 2007a), rainfall amount (Gordon, 1948; Levine and Andersen, 1973) and evaporation rate (O’Connor et al., 2008) are important regulators, and thus predictors, of

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moisture in the faeces following deposition. Availability of moisture in the faeces can also be measured directly to provide faecal moisture (FM) and O’Connor et al. (2006) have suggested that measurement of this variable is likely to integrate all of the other moisture-mediated influences on development to L3. In a prediction model for Teladorsagia (Ostertagia) and Trichostrongylus spp. developed by Callinan et al. (1982), soil moisture was one of the most important factors for successful development to L3. This is in agreement with the earlier work of Levine and Todd (1975) who suggested that temperature and soil moisture are the most important factors affecting development and survival of H. contortus L3 as soil moisture is influenced by the interaction of rainfall, soil type and evapotranspiration. While the importance of soil moisture as a moisture regulator for development of eggs to L3 on pasture has been established (Bullick and Andersen, 1978; Levine and Todd, 1975), the effect of varying soil moisture and the interactions between soil moisture, rainfall timing and rainfall amount on FM and subsequent development of H. contortus and T. colubriformis to L3 have not been investigated. In fact, little consideration has been given to soil moisture in recent ecological studies on the effect of simulated rainfall (O’Connor et al., 2007a, 2007b, 2008; Khadijah et al., 2013) and herbage height (Sakwa et al., 2003) on the development of H. contortus to L3. To investigate this, two experiments were conducted in climate-controlled chambers to determine the effect of soil moisture, rainfall timing and rainfall amount on FM and development of H. contortus and T. colubriformis to L3. A number of general hypotheses to confirm established understanding about the development of H. contortus and T. colubriformis from egg to L3 were tested but the specific hypotheses of particular interest in these experiments were (i) the recovery of L3 will increase with increasing soil moisture at the time of faecal deposition; (ii) increasing initial soil moisture will reduce the beneficial effects of increased rainfall amount on L3 recovery; (iii) FM during the first few days post deposition will be an accurate predictor of development to L3 when temperature is not limiting.

2.1.1. Experiment 1 This was a 3 × 3 × 3 incomplete factorial experiment with a completely randomised design and 4 replicates per treatment combination. The factors and levels of rainfall of either 0 mm, 12 mm or 24 mm, timed to occur on days −1, 0 or 3 relative to faecal deposition, were applied to experimental units containing soil with 0, 10 or 15% (w/w) moisture content. The design was incomplete because of the absence of 0 mm rainfall × rainfall timing. Soil was oven dried at 80 ◦ C prior to the experiment and water was added (w/w) to the respective experimental units on day −1 to attain soil moisture 10 or 15%. Recovery of L3 from faecal pellets and the top 25 mm soil was determined on day 14 after faecal deposition.

2. Material and methods

2.4. Production of infective faeces

2.1. Experimental designs

Groups (n = 8 animals/group) of Merino wethers (5–6 months old) were used in Experiments 1 and 2 as faecal donors. Following arrival at the animal house, they were weighed and treated with albendazole and levamisole (Rotate® , Novartis Animal Health Australia), naphthalaphos (Rametin® , Bayer Australia) and abamectin (Virbamec LV® , Virbac Australia) at recommended oral dose rates to remove any existing GIN infection. Nematode worm egg counts (WEC) and coproculture were negative for all animals ten days after treatment. The animals were fed daily with chaffed lucerne (Medicago sativa; 150 g/d) and oaten (Avena sativa; 600 g/d) hay, had free access to water and were kept in individual pens under natural light.

Two experiments were conducted in climate-controlled chambers to test these hypotheses. Experiment 1 was designed to determine the effects of initial soil moisture, simulated rainfall timing and simulated rainfall amount on the development of H. contortus and T. colubriformis to L3. Experiment 2 was designed to determine the effects of these factors on FM and soil moisture. In both experiments simulated rainfall (hereafter referred to as rainfall) was applied to freshly collected sheep faeces which had been placed on the surface of experimental containers filled with oven-sterilised soil.

2.1.2. Experiment 2 The design was identical to that described for Experiment 1. However, rather than measuring development to L3, faeces and soil were subsampled from experimental units on days 1, 2, 3, 4, 7, and 14 relative to faecal deposition to determine moisture content. 2.2. Experimental units Development from egg to L3 occurred in experimental units comprising polycarbonate jars (250 ml, 100 mm height, 60 mm diameter) filled with a mixture (ratio of 5:1) of sterilised sandy loam soil and 7 mm (diameter) gravel to a depth of 60 mm. Each jar had a single 2 mm drainage hole drilled in the side wall, 25 mm from the bottom of the jar. 2.3. Rainfall simulation Rainfall was applied to the experimental units via a second polycarbonate jar (250 ml, 100 mm height, 60 mm diameter) with seven holes (5.5 mm diameter) drilled into the base which was then lined with a single layer of filter paper (No. 42, Whatman® Schleicher & Schuell, England). Water was applied to experimental units by placing the rainfall jar with the required volume of water on the top of them (i.e. 40 mm above the soil surface). The rainfall jars remained in position for 6 h but the average rate of application was 24 mm/h. Rainfall of 12 and 24 mm required a volume of 34 and 68 ml respectively.

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Sheep used in Experiment 1 were orally infected with 5000 H. contortus (Kirby strain, CSIRO Livestock Industries) and 10,000 T. colubriformis (McMaster strain, CSIRO Livestock Industries) 14 days after receiving their last drench. Each animal was given an intramuscular injection of 0.5 ml Ilium Trimedexil® (5 mg/mL Dexamethasone Trimethylacetate) 24 h prior to infection. Animals used in Experiment 2 remained uninfected. Fourteen days after infection (Experiment 1) or drenching (Experiment 2), animals were allowed to graze on pastures for two weeks to enable collection of faeces representative of animals consuming fresh forage. On the last day of the grazing period, faecal bags were attached to all animals for 24 h to collect faeces for the experiments. For animals in Experiment 1, WEC and L3 differentiation were determined on day 21 post infection to confirm establishment of the infection. Fresh faeces from donor animals were gently and thoroughly mixed by hand in a large container prior to subsampling for experimental units. Faecal pellets (14.0 ± 0.2 g; approximately 24 pellets) were deposited on experimental units in an uncompacted mound to mimic field deposition. 2.5. Measurements 2.5.1. Temperature and evaporation Experiments 1 and 2 were conducted in climatecontrolled chambers (SANYO Electric Biomedical Co., Ltd., Japan). The temperature throughout all experiments was set to the long term (year 1995–2009) minimum and maximum temperatures for Armidale NSW, Australia for the summer month of February (Bureau of Meteorology). The temperatures were programmed using CALgrafix Standard 1.1.04 software (CALControls Inc., United Kingdom) attached to the climate-controlled chambers and were ramped consistently day to day throughout the experiment. The actual temperatures were recorded at 1 h intervals using Tinytag® climate data loggers (Gemini Data Loggers, Chichester, UK) located on each of the 4 shelves in the climate chamber. Evaporation rates for Experiments 1 and 2 were measured by placing a 250 ml polycarbonate jar filled with water on each shelf in the climate chamber. Losses of water were measured daily and recorded in mm/day. The climate chambers were unlit but exposed (through a clear panel in the front door) to natural lighting conditions in the laboratory. 2.5.2. Faecal moisture Faecal moisture (FM) was determined prior to deposition on experimental units for both experiments. In Experiment 2, 3–5 faecal pellets were subsampled on days 1, 2, 3, 4, 7, and 14 relative to faecal deposition. Faecal pellets were weighed and then dried at 80 ◦ C for 5 days. The samples were then reweighed to determine moisture loss and calculate FM (%). FM was not determined in Experiment 1. 2.5.3. Soil moisture In Experiment 2, soil was subsampled using a spatula (circa 2 cm3 ) on days 1, 2, 3, 4, 7 and 14 relative to faecal deposition to determine soil moisture. Soil was weighed

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and then dried at 80 ◦ C for 5 days. The samples were then reweighed to determine moisture loss and calculate soil moisture (%). 2.5.4. Parasitological methods (Experiment 1) WEC was estimated using a modified McMaster method (Whitlock, 1948) with 1 egg equivalent to 60 eggs per gram (epg) of faeces. Coproculture (MAFF, 1986) and species differentiation of L3 were conducted using the identification keys of Whitlock (1960). Intra-pellet enumeration of free-living nematodes at different stages was conducted on faecal pellets on day 14 after faecal deposition. Faecal pellets were weighed and distilled water added to provide a dilution of 22-fold. The samples were then thoroughly mixed and filtered through a 600 ␮m sieve and 60 ␮l of the filtrate was dispensed on etched glass slides with 2 drops of Lugol’s iodine. Degenerate and embryonated eggs, L1–L2 and L3 were identified as described by O’Connor (2007) and counted under 40× magnification. Extra-pellet nematode enumeration of L3 was conducted on day 14 from the top 25 mm of soil using a modified method of O’Connor et al. (2008), where subsamples were quantified by weight rather than volume. In brief, we subsampled approximately 5 g of the soil and L3 were floated by adding potassium iodide (r.d. 1.4) to the soil. The L3 were then collected using a sedimentation method and counted under 40× magnification. Prior to the experiment, validation of this method indicated a recovery of added L3 of 83% (coefficient of variation = 10%). 2.6. Statistical analysis Analysis of variance (ANOVA) was used to test the effects of experimental factors on response variables (JMP 9.0, SAS Institute Inc. 2010). The response variables were FM, soil moisture, recovery of total L3 (intra- plus extrapellet), intra-pellet L3, extra-pellet L3, pre-infective larval stages (L1–L2), embryonated eggs and degenerate eggs. Recoveries of free-living nematode stages were expressed as a percentage of the total number of eggs deposited per experimental units. The effect variables were initial soil moisture, rainfall timing, rainfall amount and collection day (for Experiment 2). Worm species was included as an effect in the model for analysis of total L3, intra-pellet L3 and extra-pellet L3 because H. contortus and T. colubriformis L3 could be reliably identified. Unwatered controls (i.e. rainfall amount = 0 mm) were excluded from the model when rainfall timing was tested because it would have prevented the testing of interactions among effects (i.e. controls did not have variable days of application). Rainfall timing was excluded from the model when testing the effects of rainfall amount and initial soil moisture so as to include unwatered controls in the analysis. Mean separation of significant (P < 0.05) effects was by protected studentized t-tests. Variables that were not normally distributed were subjected to transformation prior to analysis. The effectiveness of each transformation was confirmed on the transformed data and on the model residuals by reference to the Shapiro-Wilks statistic. Backtransformed least squares means ± 95% confidence limits

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(c.l.) are presented in the results. In the case of datasets that did not require transformation prior to statistical analysis, least squares mean ± standard errors (s.e.) are presented. For the analysis of FM and soil moisture in Experiment 2, the effects of initial soil moisture, rainfall timing, rainfall amount and collection day were fitted in a repeated measures general linear model. Significant effects (P < 0.05) were later tested with ANOVA if the interactions between treatment effects and collection day were significant. Bivariate regression analysis was conducted on untransformed data of total recovery of H. contortus and T. colubriformis L3 (Day 14, Experiment 1) with untransformed data of FM at varying times (days 1, 2, 3, 4, 7 and 14) relative to faecal deposition (Experiment 2) to determine the association between FM and L3 recovery. Coefficient of determination (R2 ) and probability values for each regression model are reported.

and evaporation rates for Experiments 1 and 2 were 1.0 and 0.93 respectively. 3.1. Experiment 1 3.1.1. Recovery of L3 A summary of the statistical significance of main treatment effects for development to various development stages is provided in Table 2. 3.1.1.1. Effect of worm species. There was higher recovery of H. contortus L3 from intra-pellet (0.8%; P < 0.001), extra-pellet (2.1%; P < 0.001) and therefore total (intra and extra-pellet) (3.7%; P < 0.001) than T. colubriformis L3 (intra-pellet L3 = 0%, extra-pellet L3 = 0.2%, total L3 = 0.4%), when averaged across the main effects of initial soil moisture, rainfall timing and rainfall amount.

3. Results Daily temperatures during the two experiments ranged from 15.7 to 33.0 ◦ C (mean of 21.8 ◦ C) in an approximately sinusoidal diurnal pattern. The evaporation rates for the two experiments ranged from 2.1 to 7.1 mm/day (mean of 3.7 mm/day). FM on the day of deposition for Experiments 1 and 2 was 69.5 and 51.8% respectively while WEC on the day of deposition for Experiment 1 was 3800 epg (Table 1). L3 differential from coproculture indicated 78% H. contortus and 22% T. colubriformis on the day of deposition. The correlation (r) values between daily temperatures

3.1.1.2. Effect of initial soil moisture. There was higher recovery of total L3 (P = 0.002) and extra-pellet L3 (P < 0.001) from initial soil moisture levels of 10 (total L3 = 2.2%; extra-pellet = 1.4%) and 15% (total L3 = 2.1%; extra-pellet = 1.1%) when compared to 0% (total L3 = 0.6%; extra-pellet L3 = 0.2%) but initial soil moisture had no effect on the recovery of intra-pellet L3 (overall mean = 0.3%; P = 0.349). There was no interaction between initial soil moisture and worm species (Table 2) on the recovery of intra-pellet L3 (Fig. 1a), extra-pellet L3 (Fig. 1b) or total L3 (Fig. 1c).

Table 1 Minimum, maximum and mean daily temperatures and evaporation rates; worm egg count (WEC; epg) on the day of deposition; and initial faecal moisture (FM; %) for Experiments 1 and 2 (means ± s.e.). Experiment

Temperature (◦ C) Min

1 2

Max

15.9 ± 0.11 33.0 ± 0.07 15.7 ± 0.13 32.9 ± 0.08

Evaporation rate (mm/day) Mean

Min

21.7 ± 0.14 21.8 ± 0.15

2.4 ± 0.18 7.1 ± 0.32 2.1 ± 0.18 6.2 ± 0.42

Max

WEC (epg)

FM (%)

3800 ± 520 n.a.

69.5 ± 0.87 51.8 ± 0.35

Mean 3.8 ± 0.27 3.6 ± 0.23

n.a.: not applicable.

Fig. 1. Main effect of initial soil moisture (0, 10 and 15%) on recovery of intra-pellet (a), extra-pellet (b) and total (intra- plus extra-pellet) (c) third stage larvae (L3) of H. contortus and T. colubriformis (back-transformed least squares mean (%) ±95% c.l.).

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Table 2 Significance of main treatment effects on the recovery of H. contortus and T. colubriformis at different developmental stages. Main treatment effects

Initial soil moisture Rainfall timing Rainfall amount Species Replicate Initial soil moisture × rainfall timing Initial soil moisture × rainfall amount Rainfall timing × rainfall amount Initial soil moisture × species Rainfall timing × species Rainfall amount × species

P values for the main treatment effects Intra-pellet L3

Extra-pellet L3

Total L3

L1-L2

Embryonated eggs

Degenerate eggs

0.349 <0.001 <0.001 <0.001 <0.001 0.101 0.026 0.163 0.168 0.433 0.793

<0.001 <0.001 <0.001 <0.001 <0.001 0.374 0.157 0.069 0.181 0.412 0.049

0.002 <0.001 <0.001 <0.001 <0.001 0.706 0.023 0.247 0.080 0.638 0.224

0.984 0.028 0.250 n.a. <0.001 0.839 0.890 0.316 n.a. n.a. n.a.

0.205 0.430 0.040 n.a. <0.001 0.858 0.566 0.356 n.a. n.a. n.a.

0.996 0.274 0.203 n.a. 0.005 0.206 0.569 0.667 n.a. n.a. n.a.

n.a.: not applicable because species could not be reliably identified to allow differentiation.

3.1.1.3. Effect of rainfall timing. There was higher recovery of intra-pellet L3 (P < 0.001) as result of rainfall on days −1 (0.6%) and 0 (1.2%) relative to faecal deposition when compared to rainfall on day 3 (0.1%). There was higher recovery of total L3 (8.3%, P < 0.001) and extra-pellet L3 (5.6%; P < 0.001) from rainfall on day 0 when compared to other rainfall days. There was no significant interaction between the effects of initial soil moisture and rainfall timing on the recovery of intra-pellet L3, extra-pellet L3 or total L3 (Table 2). Similarly, there was no significant interaction between the effect of rainfall timing and worm species (Table 2) on the recovery of intra-pellet L3 (Fig. 2a), extra-pellet L3 (Fig. 2b) and total L3 (Fig. 2c). 3.1.1.4. Effect of rainfall amount. There were higher recoveries of total L3 (6.0%; P < 0.001) and extra-pellet L3 (3.7%; P < 0.001) from 24 mm of rainfall than recorded from 12 mm (total L3 = 2.5%; extra-pellet L3 = 1.4%) and 0 mm rainfall (no recovery for total and extra-pellet L3). There was no interaction between the effects of rainfall amount and worm species (Table 2) on the recovery of intra-pellet L3 (Fig. 3a) and total L3 (Fig. 3c). In contrast, there was a significant interaction between the effects of rainfall amount and worm species on the recovery of

extra-pellet L3 (P = 0.049) (Fig. 3b). The form of the interaction was such that recovery of H. contortus and T. colubriformis extra-pellet L3 was the same at 0 mm rainfall but was highest for H. contortus at 12 and 24 mm. The effect of rainfall amount on development to intrapellet (Fig. 4a) and total L3 (Fig. 4c), but not extra-pellet L3 (Fig. 4b), differed according to initial soil moisture (Table 2). When initial soil moisture was low (0%) each level of rainfall amount led to increased development of total L3. In contrast, there was no benefit for recovery of total L3 from increasing rainfall from 12 to 24 mm when initial soil moisture was 10 or 15%. There was no significant interaction between the effects of timing and amount of rainfall on the recoveries of intrapellet L3, extra-pellet L3 and total L3 (Table 2). 3.1.2. Recovery of other stages 3.1.2.1. Effect of initial soil moisture. Initial soil moisture did not affect the recovery of L1-L2 (39.3%), embryonated eggs (3.5%) or degenerate eggs (1.3%) (Table 2). 3.1.2.2. Effect of rainfall timing. There was higher recovery of L1–L2 from rainfall on day −1 relative to faecal deposition (47.7%) (P < 0.001) compared to rainfall on day 0 (31.8%) and day 3 (25.1%) There was no effect of the timing

Fig. 2. Main effect of simulated rainfall timing (days −1, 0 and 3 relative to faecal deposition) on recovery of intra-pellet (a), extra-pellet (b) and total (intra- plus extra-pellet) (c) third stage larvae (L3) of H. contortus and T. colubriformis (back-transformed least squares mean (%) ±95% c.l.).

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Fig. 3. Main effects of simulated rainfall amount (0, 12 and 24 mm) on recovery of intra-pellet (a), extra-pellet (b) and total (intra- plus extra-pellet L3) (c) third stage larvae (L3) of H. contortus and T. colubriformis (back-transformed least squares mean (%) ±95% c.l.). Means not sharing the same letter differ significantly (P < 0.05) for extra-pellet L3.

Fig. 4. Interaction between initial soil moisture (0, 10 and 15%) and simulated rainfall amount (0, 12 and 24 mm) on recovery of intra-pellet (a), extra-pellet (b) and total (c) third stage larvae (L3) of H. contortus and T. colubriformis (back-transformed least squares means (%) ±95% c.l.). Means not sharing the same letter differ significantly for intra-pellet L3 (P < 0.05) and extra-pellet L3 (P < 0.05).

of rainfall on the recovery of embryonated (2.6%) or degenerate eggs (1.3%) and neither was the interaction between the effects of initial soil moisture and rainfall timing significant for recovery of L1–L2, embryonated eggs or degenerate eggs (Table 2). Larval differentiation of L3 from coproculture of faeces collected on day 14 (Table 3) indicated a dominance of T. colubriformis with the exception of faeces which had rainfall on the day of deposition.

3.1.2.3. Effect of rainfall amount. There was no significant effect of rainfall amount on the recovery of L1–L2 (39.8%) or degenerate eggs (1.3%). In contrast, there was a significant effect of rainfall amount on the recovery of embryonated eggs (P = 0.040) with recovery being 5.6% from 0 mm rainfall treatment and 1.8% and 3.4% from 12 and 24 mm rainfall treatments respectively. 3.2. Experiment 2

Table 3 Species of third stage infective larvae (L3) (%) from coproculture of faeces collected on day 14 after receiving rainfall on days −1, 0 or 3 in relation to faecal deposition. Unwatered controls are included for comparative purposes. Rainfall day

H. contortus (%)

T. colubriformis (%)

Control −1 0 3

33 43 64 41

67 57 36 59

3.2.1. Faecal moisture A summary of the statistical significance of main treatment effects for FM and soil moisture is provided in Table 4. 3.2.1.1. Effect of initial soil moisture. Mean FM (across all collection days) was highest (P = 0.004) when initial soil moisture was 10 and 15%. There was no significant interaction between the effects of initial soil moisture and collection day (Table 4) on FM, but there was a trend that

S. Khadijah et al. / Veterinary Parasitology 196 (2013) 161–171 Table 4 Significance of main treatment effects on moisture content of faecal pellets and soil when averaged across collection days. Main treatment effects

P value

Initial soil moisture Collection day Rainfall timing Rainfall amount Initial soil moisture × collection day Initial soil moisture × rainfall timing Initial soil moisture xrainfall amount Rainfall timing × collection day Rainfall amount × collection day Rainfall timing × rainfall amount

Faecal moisture

Soil moisture

0.004 <0.001 <0.001 <0.001 0.101 0.202 0.516 <0.001 0.005 0.015

<0.001 <0.001 <0.001 <0.001 0.251 0.463 <0.001 <0.001 0.002 0.263

FM declined most rapidly from treatments with 0% initial soil moisture (Table 5). FM of unwatered controls with initial soil moisture of 0% declined from 58.1% on the day of deposition to 8.9% by day 14 relative to faecal deposition. 3.2.1.2. Effect of rainfall timing. Mean FM (across all collection days) was greatest when rainfall occurred on the day of deposition (day 0; P < 0.001). There was an interaction between the effect of rainfall timing and collection day (P < 0.001). The form of this interaction was such that rainfall on day 0 led to higher FM on days 1, 2 and 3 after faecal deposition compared to all other treatments. From day 4 post deposition, differences in FM among rainfall days began to diminish (Table 6). 3.2.1.3. Effect of rainfall amount. FM was highest from rainfall amounts of 12 and 24 mm when averaged over collection days (P < 0.001). There was a significant interaction between the effects of rainfall amount and collection day on FM (P = 0.005). The form of interaction was such that rainfall did not significantly increase FM until day 2 and was higher from 24 mm on days 4 and 7 (Table 7). There was no significant interaction between the effects of initial soil moisture and rainfall amount on FM (Table 4). There was an interaction between the effect of rainfall amount and rainfall timing (P = 0.015) on FM across all collection days, such that the highest FM was from 24 mm rainfall simulated on day 1 relative to faecal deposition. 3.2.2. Soil moisture 3.2.2.1. Effect of initial soil moisture. Mean soil moisture averaged across all collection days was greatest (P < 0.001)

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when initial soil moisture was 10 or 15% as compared to 0%. Initial soil moisture of 10% and 15% led to higher soil moisture on days 0, 1, 2, 4 and 7 after faecal deposition when averaged across rainfall timing and rainfall amount (Table 8). The soil moisture content of unwatered controls with initial soil moisture of 0% ranged from 0.7–1.7% during the 14-day period of this experiment. There was an interaction between the effect of initial soil moisture and rainfall amount on soil moisture across all collection days (P < 0.001), such that an increase of rainfall amount gave more advantage when initial soil moisture was 0% and 10% compared to 15%. 3.2.2.2. Effect of rainfall timing. Soil moisture content resulting from rainfall on days −1 and 0 relative to faecal deposition was similar across all days and greater than rainfall on day 3 until day 4 post deposition (Table 9). 3.2.2.3. Effect of rainfall amount. Soil moisture content increased with each level of rainfall amount when averaged across collection days (P < 0.001). The interaction between rainfall amount and collection day was significant (Table 4) for soil moisture content because differences as a result of 12 and 24 mm took a few days to become apparent (Table 10). 3.2.3. Relationship between total L3 recovery (Experiment 1) and faecal moisture (Experiment 2) Coefficient of determination (R2 ) and probability values (P) from the regression between total recovery of H. contortus and T. colubriformis L3 at day 14 with FM at days 0, 1, 2, 3, 4, 7 and 14 after faecal deposition are reported in Table 11. The relationship between FM and L3 recovery for both worm species was strongest for day 3 FM (Fig. 5a and b, respectively). 4. Discussion Mean daily minimum and maximum temperatures in the two experiments were higher than the long term values (1995–2009) for Armidale (12.9 and 25.1 ◦ C respectively) (Bureau of Meteorology) and exceeded the minimum temperature requirements for development of H. contortus and T. colubriformis (Barger et al., 1974). Mean daily evaporation rate was lower than the long term value of 4.4 mm/day. FM on the day of deposition (69.5%) for Experiment 1 was comparable to the optimum FM values required for the development of H. contortus (70%) and T. colubriformis

Table 5 Moisture content (least squares means (%) ± s.e.) of faecal samples, collected at varying days relative to faecal deposition, deposited on soil with initial moisture content of 0, 10 or 15% (averaged across rainfall timing and amount). Means within each column not sharing the same letter differ significantly (P = 0.036). Initial soil moisture (%)

0 10 15 P n.a.: not applicable.

Faecal moisture (FM, %) at collection days relative to faecal deposition 0

1

2

3

4

7

14

58.1 ± 0.4 58.1 ± 0.4 58.1 ± 0.4 n.a.

34.5 ± 2.3 40.2 ± 2.3 34.5 ± 2.3 0.138

20.8 ± 3.0 29.9 ± 3.0 25.6 ± 3.0 0.108

15.3 ± 2.1 20.1 ± 2.1 19.4 ± 2.1 0.213

21.5 ± 2.2 26.9 ± 2.2 28.3 ± 2.2 0.069

14.5b ± 1.7 19.8a ± 1.7 20.0a ± 1.7 0.036

11.0 ± 1.3 10.8 ± 1.3 13.7 ± 1.3 0.219

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Table 6 Moisture content (least squares means (%) ± s.e.) of faecal samples collected at varying days relative to faecal deposition, which had received rainfall on day -1, 0 or 3 (averaged across initial soil moisture and rainfall amount). Means within each column not sharing the same letter differ significantly. Rainfall timing (day)

−1 0 3 P

Faecal moisture (FM, %) at collection days relative to faecal deposition 0

1

2

3

4

7

14

58.1 ± 0.4 58.1 ± 0.4 58.1 ± 0.4 n.a.

36.3b ± 1.7 47.7a ± 1.7 30.2c ± 1.7 <0.001

33.0b ± 2.0 40.2a ± 2.0 15.9c ± 2.0 <0.001

22.3b ± 1.4 30.0a ± 1.4 12.3c ± 1.4 <0.001

27.2b ± 2.1 34.5a ± 2.1 33.5a ± 2.1 0.035

20.7 ± 1.6 22.3 ± 1.6 22.4 ± 1.6 0.689

10.6 ± 1.2 12.7 ± 1.2 14.5 ± 1.2 0.082

n.a.: not applicable. Table 7 Moisture content (least squares means (%) ± s.e.) of faecal samples collected at varying days relative to faecal deposition, which received 0 (unwatered controls), 12 or 24 mm rainfall (averaged across rainfall timing and initial soil moisture). Means within each column not sharing the same letter differ significantly. Rainfall amount (mm)

0 12 24 P

Faecal moisture (FM, %) at collection days relative to faecal deposition 0

1

2

3

4

7

14

58.1 ± 0.4 58.1 ± 0.4 58.1 ± 0.4 n.a.

33.0 ± 3.1 37.8 ± 1.8 38.4 ± 1.8 0.317

17.0b ± 4.1 29.4a ± 2.3 30.0a ± 2.3 0.019

11.8b ± 2.8 20.6a ± 1.6 22.5a ± 1.6 <0.001

13.3c ± 3.0 28.8b ± 1.7 34.7a ± 1.7 <0.001

10.6c ± 2.3 18.5b ± 1.3 25.2a ± 1.3 <0.001

10.3 ± 1.7 11.7 ± 1.0 13.5 ± 1.0 0.208

n.a.: not applicable. Table 8 Moisture content (least squares means (%) ± s.e.) of soil collected at varying days relative to faecal deposition, where initial moisture levels were 0, 10 or 15% (averaged across rainfall amount and timing). Means within each column not sharing the same letter differ significantly. Initial soil moisture (%)

0 10 15 P

Soil moisture (%) at collection days relative to faecal deposition −1

0

1

2

3

4

7

14

0.7 ± 0 10 ± 0 15 ± 0 n.a.

4.1b ± 1.4* 7.6ab ± 1.4 10.3a ± 1.4 0.011

7.6b ± 1.4** 11.2ab ± 1.4 12.8a ± 1.4 0.034

6.8b ± 1.4 10.5ab ± 1.4 11.4a ± 1.4 0.042

6.1 ± 1.3 9.0 ± 1.3 9.2 ± 1.3 0.163

9.9b ± 0.5*** 12.9a ± 0.5 13.1a ± 0.5 <0.001

7.9b ± 0.6 10.2a ± 0.6 10.1a ± 0.6 0.017

2.7 ± 0.5 3.0 ± 0.5 3.5 ± 0.5 0.546

Soil moisture rise because of the inclusion of rainfall on days −1 (*), 0 (**) and 3 (***) relative to faecal deposition.

(55–60%) eggs to L3 in the studies by Rossanigo and Gruner (1995). FM on the day of deposition for Experiment 2 was lower than the value in Experiment 1. We observed higher recovery of total L3 with initial soil moisture values of 10 and 15% compared with dry soil. Higher initial soil moisture also resulted in higher FM and

soil moisture values. It is likely that the benefit of initial soil moisture for L3 development was mediated by increased FM. This is supported by the significant and positive relationship between FM in Experiment 2 and total L3 recovery in Experiment 1 which was strongest for FM on days 1–3 after faecal deposition. Levine and Todd (1975) suggested

Table 9 Moisture content (least squares means (%) ± s.e.) of soil collected at varying days and which received rainfall on days −1, 0 or 3 relative to faecal deposition (averaged across initial soil moisture and rainfall amount). Means within each column not sharing the same letter differ significantly. Rainfall timing (day)

−1 0 3 P

Soil moisture (%) at collection days relative to faecal deposition 0

1

2

3

4

7

14

17.8a ± 0.3 4.3b ± 0.3 4.5b ± 0.3 <0.001

17.6a ± 0.5 17.4a ± 0.5 4.6b ± 0.3 <0.001

15.8a ± 0.5 16.9a ± 0.5 4.0b ± 0.5 <0.001

14.0a ± 0.5 14.9a ± 0.5 3.3b ± 0.5 <0.001

15.4b ± 0.6 15.5b ± 0.6 17.5a ± 0.6 0.039

11.3b ± 0.7 13.0ab ± 0.7 14.3a ± 0.7 0.013

2.5b ± 0.5 4.2a ± 0.5 4.5a ± 0.5 0.022

Table 10 Moisture content (least squares means (%) ± s.e.) of soil collected at varying days at various timesafter faecal depositionwhich received rainfall of 0, 12 or 24 mm (averaged across initial soil moisture and rainfall timing). Means within each column not sharing the same letter differ significantly. Rainfall amount (mm)

Soil moisture (%) at collection days relative to faecal deposition 0

0 12 24 P

1

4.2 ± 1.9 8.1ab ± 1.1 9.7a ± 1.1 0.050 b

2

5.2 ± 1.9 12.0a ± 1.1 14.4a ± 1.1 <0.001 b

3

4.2 ± 1.8 10.5b ± 1.0 14.0a ± 1.0 <0.001 c

4

2.8 ± 1.7 8.5b ± 1.0 13.0a ± 1.0 <0.001 c

7

3.6 ± 0.7 13.8b ± 0.4 18.4a ± 0.4 <0.001 c

14

2.5 ± 0.8 10.3b ± 0.5 15.4a ± 0.5 <0.001 c

1.7b ± 0.7 3.2ab ± 0.4 4.3a ± 0.4 0.009

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Fig. 5. Bivariate regression with linear fit of total third stage larvae (L3) recovery (%; raw data) of H. contortus (a) and T. colubriformis (b) with faecal moisture (FM %; raw data) at day 3 relative to faecal deposition. Recovery of total H. contortus L3 = −4.03 + 0.77 × FM on day 3 relative to faecal deposition (R2 = 0.46; P < 0.001). Recovery of total T. colubriformis L3 = −2.21 + 0.29 × FM on day 3 relative to faecal deposition (R2 = 0.32; P < 0.001).

that temperature and soil moisture are the most important factors affecting development and survival of H. contortus, as soil moisture reduces the temperature at the ground surface and provides moisture to the grass that will reduce the drying rate of faecal pellets (Bullick and Andersen, 1978). SM values differ between soil types, increasing with clay content, and at field capacity can range from 15 to 45% (Rab et al., 2011). A SM value of 15% was the upper limit for the sandy loam soil used in these experiments to avoid saturation following application of rainfall. Our hypothesis that higher initial soil moisture would reduce the degree by which higher rainfall amounts increased the recovery of H. contortus and T. colubriformis L3 was supported by the data. Increasing the amount of rainfall from 12 to 24 mm led to greater recovery of total L3 when initial soil moisture was 0%, but had no effect on total L3 recovery when initial soil moisture was 10 or 15% as sufficient moisture for development was already present in the pellets. This trend was also observed for soil moisture content and FM. When initial soil moisture was 0%, increasing rainfall from 12 to 24 mm increased mean soil moisture content from 5.7 to 12.2% and FM from 20.2 to 24.4%. In contrast, when initial soil moisture was 15% increasing rainfall only increased mean soil moisture content from 12.0 to 12.7% and did not increase FM (27.2% for 12 and 24 mm rainfall). It is likely that maximum values for soil moisture content were attained by the application of 24 mm rainfall, as maximum mean soil moisture values did not exceed Table 11 Coefficient of determination (R2 ) and probability values (P) from the regression between total recovery of third stage larvae (L3) at day 14 of H. contortus and T. colubriformis and moisture content of faecal pellets collected on days 1, 2, 3, 4, 7 and 14 after faecal deposition. Collection day

Total H. contortus L3 2

1 2 3 4 7 14

Total T. colubriformis L3

R

P

R2

P

0.34 0.41 0.46 0.26 0.36 0.17

<0.001 <0.001 <0.001 <0.001 <0.001 <0.001

0.24 0.25 0.32 0.16 0.21 0.11

<0.001 <0.001 <0.001 <0.001 <0.001 0.002

15–17%, and this may account for greater effects of rainfall when initial soil moisture was lower. The benefit of rainfall amount on L3 development in a dry environment was shown by Agyei (1997) who reported a high correlation between rainfall amount and recovery of L3 from pasture. It is clear from our results that in a controlled environment, initial soil moisture level can regulate the effect of rainfall on development to L3 by providing an alternative source of moisture for faecal pellets. This confirms the importance of including soil moisture in prediction models of GIN development (Callinan et al., 1982; Levine and Todd, 1975), but further confirmation with field studies is required. Earlier observations of the benefit of rainfall amount on development success have not always included a range of initial soil moisture levels (e.g. O’Connor et al., 2007a) nor reported initial values. Where rainfall amount has been tested at low initial soil moisture values (say lower than 10%) it is likely that the magnitude of the benefit of the applied rainfall for L3 development may be considerably more than if soils of higher moisture content had been used. FM three days after faecal deposition was best associated with the total recovery of H. contortus and T. colubriformis L3 but FM values on days 1 and 2 also provided good predictive value. This confirms that FM close to faecal deposition is a good predictor of successful development of H. contortus and T. colubriformis to L3. O’Connor et al. (2007b) had suggested FM on day 4 relative to faecal deposition was the best predictor for extra-pellet recovery of H. contortus L3 but those authors did not measure FM closer to the day of deposition. Khadijah et al. (2013) suggested that development success of H. contortus and T. colubriformis was best predicted from FM on days 2 and 3, and days 3 and 4, respectively. The consistency of these observations suggests that FM within 4 days of faecal deposition is an important determinant of successful development of H. contortus and T. colubriformis to L3. The regression analysis used to determine the association between FM and total L3 recovery also allowed calculation of the minimum FM value which permitted development of H. contortus and T. colubriformis to L3. Recovery of L3 occurred when FM (day 3 post deposition) exceeded 5% and 8% for H. contortus and T. colubriformis,

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respectively, and thereafter increased at the rate of 0.77 and 0.29%, respectively, for every unit increase in day 3 FM. The developmental threshold FM values are considerably lower than the minimum values suggested by Rossanigo and Gruner (1995) permissible of 1% development to L3 at a constant temperature of 23 ◦ C: 39% FM for H. contortus and 35% FM for T. colubriformis. It is likely that our approach of starting with fresh faeces (at 70% FM) and maintaining a temperature regimen with diurnal fluctuation (16–33 ◦ C) but with similar mean temperature as Rossanigo and Gruner (1995) allowed development in the few days after deposition while FM was declining and this may account for the lower estimates of FM permissible for L3 development. It is also possible that the lower initial FM in Experiment 2 (52%) resulted in an underestimate of minimum FM values permissible of development. However, we do not believe that this is likely as faecal pellets from Experiment 1 with higher FM would have experienced a greater rate of drying (Khadijah et al., 2013) such that FM by day 3 post-deposition would have been similar for faecal pellets from Experiments 1 and 2. Rainfall on the day of deposition was observed to be the most effective timing for development of extra-pellet H. contortus and T. colubriformis L3. This finding supports results by O’Connor et al. (2007a) and Khadijah et al. (2013) who suggested that rainfall which occurred within a few hours of faecal deposition would lead to the highest recovery of L3. When rainfall occurred soon after faecal deposition, FM was sustained at an elevated level for a longer period compared to when rainfall was applied on later days (Khadijah et al., 2013). Interestingly, rainfall on day 0 led to higher L3 recovery compared to rainfall on day −1, even though the soil moisture level for both rainfall days was similar for the seven days after faecal deposition. What differed between these treatments was FM, which was greater from day 1–4 when rainfall occurred on the day of deposition. It seems likely that faecal pellets exposed to the rainfall event retained more moisture (and were able to absorb more moisture from the soil) than pellets which were deposited on moist soil but not directly exposed to rainfall (e.g. day −1 rainfall), resulting in greater L3 development. We have previously reported that pellets exposed to rainfall soon after deposition have physical characteristics which are favourable for sustained changes in FM (Khadijah et al., 2013). Recovery of total H. contortus L3 on day 14 relative to faecal deposition was higher than T. colubriformis L3, indicating that H. contortus responds to additional moisture more quickly than T. colubriformis. Species composition on the day of deposition determined from coproculture was 78% H. contortus and 22% T. colubriformis, but after 14 days of experimental conditions the remaining immature developmental stages were largely dominated by T. colubriformis. The day 0 rainfall treatment was the exception, with H. contortus present in a greater percentage. This supports our understanding (Khadijah et al., 2013) that T. colubriformis is generally slower to emerge as L3 from faecal pellets (Banks et al., 1990; Gibson and Everett, 1972), instead using the pellet as a reservoir (Anderson, 1972). The presence of soil moisture in this study led to higher recovery of H. contortus L3 across all treatments (mean

recovery = 3.7%) than the earlier report of Khadijah et al. (2013; mean recovery = 0.5%) where initial soil moisture was similar to the 0% level used in the current experiment. In contrast, the recovery of T. colubriformis L3 in this paper (0.4%) was similar to the value reported by Khadijah et al. (2013; 0.4%) suggesting that availability of moisture in the soil was less beneficial for the development of T. colubriformis to L3 compared to H. contortus. The L1–L2 larvae had the highest recovery of all developmental stages and were most numerous when simulated rainfall occurred on the day prior to deposition. In contrast, Khadijah et al. (2013) reported L1–L2 to be the least numerous and degenerate eggs the most numerous stage after 14 days of experimental conditions. Higher initial soil moisture increased FMC in the current experiment which may have provided sufficient time for eggs to develop to pre-infective stages. Based on these findings we can conclude that soil moisture plays an important role in regulating the developmental success of H. contortus and T. colubriformis to L3. This role is mediated through effects on FM where higher levels in the first 3 days after faecal deposition support greater L3 development. Soil moisture also mediates the benefit of rainfall amount for L3 development, with rainfall being most beneficial on dry soils. This interaction has important consequences for epidemiological models of development and highlights the need to accommodate principal factors controlling soil moisture. The benefit obtained from rainfall falling onto the faecal pellet (i.e. on the day of faecal deposition) was confirmed but so too was the lesser, but still valuable, benefit obtained from rainfall on the day prior to deposition. It is likely that soil moisture may play an even greater role in regulating L3 development than reported here as soil moisture in the field may well be sustained for a longer period than in the experimental units used in these controlled-climate experiments. In addition, the layer of grass typically found between soil and faeces in the field would contribute to a sustained moisture environment. The aspect of sustained moisture will be the focus of further studies. Acknowledgments The principal author (Khadijah S.) was supported by a postgraduate scholarship from the Ministry of Higher Education Malaysia. The authors wish to acknowledge the skilled staff from the School of Environmental and Rural Sciences: Mr. Michael Raue and Mr. Michael Faint for technical support, and Mr. Grahame Chaffey and Mr. Paul Arnott for the assistance with the experimental animals. References Anderson, N., 1972. Trichostrongylid infections of sheep in a winter rainfall region. I. Epizootiological studies in the Western District of Victoria, 1966–67. Aust. J. Agric. Res. 23, 1113–1129. Agyei, A.D., 1997. Seasonal changes in the level of infective strongylate nematode larvae on pasture in the coastal savanna regions of Ghana. Vet. Parasitol. 70, 175–182. Barger, I.A., Benyon, P.R., Southcott, W.H., 1974. Simulation of larval populations of a parasitic nematodes of sheep. Simulation 22, 81–84. Banks, D.J.D., Singh, R., Barger, I.A., Pratap, B., Le Jambre, L.F., 1990. Development and survival of infective larvae of Haemonchus contortus and

S. Khadijah et al. / Veterinary Parasitology 196 (2013) 161–171 Trichostrongylus colubriformis on pasture in a tropical environment. Int. J. Parasitol. 20 (2), 155–160. Barnes, E.H., Dobson, R.J., Donald, A.D., Waller, P.J., 1988. Predicting populations of Trichostrongylus colubriformis infective larvae on pasture from meteorological data. Int. J. Parasitol. 18 (6), 767–774. Besier, R.B., Dunsmore, J.D., 1993. The ecology of Haemonchus contortus in a winter rainfall region in Australia – the development of eggs to infective larvae. Vet. Parasitol. 45 (3/4), 275–292. Bullick, G.R., Andersen, F.L., 1978. Effects of irrigation on survival of thirdstage Haemonchus contortus larvae (Nematoda: Trichostrongylidae). The Great Basin Naturalist. 38 (4), 369–378. Bureau of Meteorology, Commonwealth of Australia, 2010. Climate Data Online. Retrieved from/(accessed 01.03.10). Callinan, A.P.L., Morley, F.H.W., Arundel, J.H., White, D.H., 1982. A model of the life cycle of sheep nematodes and the epidemiology of nematodiasis in sheep. Agric. Sys. 9, 199–225. Gibson, T.E., Everett, G., 1972. The ecology of the free-living stages of Ostertagia circumcincta. Parasitology 64, 451–460. Gordon, H.M., 1948. The epidemiology of parasitic diseases, with special reference to studies with nematode parasites of sheep. Aust. Vet. J. 24, 17–45. Khadijah, S., Kahn, L.P., Walkden-Brown, S.W., Bailey, J.N., Bowers, S.F., 2013. Effect of simulated rainfall timing on faecal moisture and development of Haemonchus contortus and Trichostrongylus colubriformis eggs to infective larvae. Vet. Parasitol. 192 (1–3), 199–210. Levine, N.D., Andersen, F.L., 1973. Development and survival of Trichostrongylus colubriformis on pasture. J. Parasitol. 59 (1), 147–165. Levine, N.D., Todd Jr., K.S., 1975. Micrometeorological factors involved in development and survival of free-living stages of the sheep nematodes Haemonchus contortus and Trichostrongylus colubriformis. A review. Int. J. Biometeorol. 19 (3), 174–183. MAFF (Ed.), 1986. Manual of Veterinary Parasitological Laboratory Techniques. Ministry of Agriculture, Fisheries and Food/HMSO, London, UK. O’Connor, L.J., 2007. Ecology of the free-living stages of Haemonchus contortus in a cool temperate environment, PhD. School of Rural Science and Agriculture, Armidale, University of New England, 158 pp.

171

O’Connor, L.J., Kahn, L.P., Walkden-Brown, S.W., 2007a. The effects of amount, timing and distribution of simulated rainfall on the development of Haemonchus contortus to the infective larval stage. Vet. Parasitol. 146 (1/2), 90–101. O’Connor, L.J., Kahn, L.P., Walkden-Brown, S.W., 2007b. Moisture requirements for the free-living development of Haemonchus contortus: quantitative and temporal effects under conditions of low evaporation. Vet. Parasitol. 150 (1/2), 128–138. O’Connor, L.J., Kahn, L.P., Walkden-Brown, S.W., 2008. Interaction between the effects of evaporation rate and amount of simulated rainfall on development of the free-living stages of Haemonchus contortus. Vet. Parasitol. 155 (3/4), 223–234. O’Connor, L.J., Walkden- Brown, S.W., Kahn, L.P., 2006. Ecology of the free-living stages of major trichostrongylid parasites of sheep. Vet. Parasitol. 142 (1/2), 1–15. Rab, M.A., Chandra, S., Fisher, P.D., Robinson, N.J., Kitching, M., Aumann, C.D., Imhof, M., 2011. Modelling and prediction of soil water contents at field capacity and permanent wilting point of dryland cropping soils. Soil Res. 49, 389–407. Rossanigo, C.E., Gruner, L., 1995. Moisture and temperature requirements in faeces for the development of free-living stages of gastrointestinal nematodes of sheep, cattle and deer. J. Helminthol. 69 (4), 357–362. Sakwa, D.P., Walkden-Brown, S.W., Dobson, R.J., Kahn, L.P., Lea, J.M., Baillie, N.D., 2003. Pasture microclimate can influence early development of Haemonchus contortus in sheep faecal pellets in a cool temperate climate. In: The Australian Sheep Veterinary Society, Cairns, May 25–30, pp. 33–44. Waller, P.J., 1971. Structural differences in the egg envelopes of Haemonchus contortus and Trichostrongylus colubriformis (Nematoda: Trichostrongylidae). Parasitology 62, 157–160. Whitlock, H.V., 1948. Some modifications of the McMaster helminth egg-counting technique and apparatus. J. Counc. Sci. Ind. Res. 21, 177–180. Whitlock, H.V., 1960. The differentiation of the immature forms of rhabditida of domestic animals. In: Veterinary Helminthology. Lea & Febiger, Philadelphia, pp. 157–172.