Moisture requirements for the migration of Haemonchus contortus third stage larvae out of faeces

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Veterinary Parasitology 204 (2014) 258–264

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Veterinary Parasitology

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Moisture requirements for the migration of Haemonchuscontortus third stage larvae out of faeces

Tong Wanga, J.A. van Wykb, A. Morrisonc, E.R. Morgana,d,∗

a School of Biological Sciences, University of Bristol, Woodland Road, Bristol BS8 1UG, UKb Department of Veterinary Tropical Diseases, University of Pretoria, Private Bag X20, Hatfield, Pretoria 0028, South Africac Moredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik, Midlothian EH26 0PZ, UKd School of Veterinary Science, University of Bristol, Langford House, Langford, Somerset BS40 5DU, UK

a r t i c l e i n f o

Article history:Received 4 January 2014Received in revised form 5 May 2014Accepted 7 May 2014

Keywords:NematodeMigrationRainfallRelative humidity (RH)Faecal moisture content (FMC)Epidemiology

a b s t r a c t

The abomasal nematode Haemonchus contortus causes severe disease and production lossin small ruminants in warmer regions and is also an emerging threat in many temperateclimates. Specific knowledge of the effects of climate on the epidemiology of H. contortusis needed to effectively apply sustainable control strategies, which rely on prediction ofinfection risk. Although the effects of temperature and rainfall on larval development inthis species have been characterised, much less is known about migration out of faecesand onto herbage. This is an important deficit in our understanding of the epidemiology ofhaemonchosis in regions with relatively low and particularly erratic rainfall. Methods weredeveloped to assess the migration of third stage larvae (L3) out of faeces under simulatedrainfall in the laboratory. These were applied in a series of experiments, which showedthat rainfall is required for migration. However, a single rainfall event was not sufficientfor migration from faeces of which the crust has hardened after having been kept in dryconditions. Light and regular rainfall resulted in rapid emergence from moist faeces kept inhumid conditions, but much slower emergence from dry faeces in dry conditions. Ambientrelative humidity therefore appears to act through faecal moisture content to modify theeffect of rainfall on larval migration. Larvae survived well in dry faeces for a number of

days, but did not migrate in the absence of rainfall, so sheep faeces could potentially actas a larval reservoir in dry conditions, with peaks of infection following rainfall. Rates offaecal desiccation and rehydration on pasture could therefore be highly relevant to temporalpatterns of larval availability.

1. Introduction

Gastrointestinal nematodes (GIN) are among the

most important pathogens of ruminants worldwide,and are responsible for considerable production lossin sheep and goats. Haemonchus contortus (superfamily

∗ Corresponding author at: School of Veterinary Sciences, University ofBristol, Langford House, Langford, Bristol BS40 5DU, UK.Tel.: +44 1179289000.

E-mail address: [email protected] (E.R. Morgan).

http://dx.doi.org/10.1016/j.vetpar.2014.05.0140304-4017/© 2014 Elsevier B.V. All rights reserved.

© 2014 Elsevier B.V. All rights reserved.

Trichostrongyloidea) can be regarded as the most impor-tant species in tropical and sub-tropical areas; in theseregions, high temperature creates favourable environ-ments for development of the free-living stages, providingsufficient moisture is available (Kao et al., 2000; O’Connoret al., 2006). As anthelmintic resistance increasingly limitsthe options for control (Papadopoulos, 2008), and climatechange appears to augment the problems caused by GIN

in temperate areas (Van Dijk et al., 2008); it is more per-tinent than ever to improve understanding of the effectof climatic conditions on larval dynamics and infectionpressure.

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Some early researchers concluded that 50 mm ofonthly rainfall was the minimum precipitation required

or outbreaks of haemonchosis (reviewed by O’Connort al., 2006). Similarly, Young (1983) reported effects ofainfall on the free-living stages of Ostertagia (Teladorsagia)p. in Australia, finding that 60 mm of rain in Februaryas sufficient to facilitate the migration of infective lar-

ae from sheep faeces to pasture herbage. However, theseesults were not specific enough to guide control strategiess they did not investigate the detailed effects of distri-ution, amount and character of rainfall on infection risk.tudies carried out by Reynecke et al. (2011) showed thatot only total amount of recorded rainfall, but also itsemporal distribution, was important in predicting devel-pmental success of H. contortus. Other workers (Bullicknd Andersen, 1978; Gruner et al., 1989) indicated thatuch higher development and survival of H. contortus

ould be achieved when enough free water was availablen the pasture. More specifically, Onyali et al. (1990) mea-ured H. contortus L3 development and survival on pasturen Nigeria, and suggested that a daily minimum rainfall of

mm was sufficient for eggs to develop to infective lar-ae and migrate onto pasture in that area. Agyei (1997)urther indicated that a minimum total amount of rainfalls required for larval availability on pasture, rather than aumber of rain days. This means that a small amount ofainfall spread over a long period might lead to little lar-al availability, compared with more intense rainfall over

shorter period, which exceeds the thresholds requiredor larval development and migration. Epidemiological pat-erns in arid areas also stress the importance of rainfall toarval availability. Jacquiet et al. (1995) studied the preva-ence of Haemonchus sp. in the Sahelian region. Here, L3vailability was limited by an extremely dry climate, withransmission only taking place after rainfall. Heavy rainfallan sometimes paradoxically lead to low larval recoverys it may wash larvae off the herbage, carry them away inhe runoff and down into the soil (Chaudary et al., 2008;tromberg, 1997).

In a study carried out by O’Connor et al. (2007), sheepaeces containing H. contortus eggs were placed in experi-

ental units containing sterile soil, and different amountsf rainfall were applied. Recovery of L3 increased withncreasing amounts of simulated rainfall and was signifi-antly higher after a single rainfall event, compared withhree smaller, split events. However, the effects of rainfalln larval development and migration were not separatedn this study.

Van Dijk and Morgan (2011) showed that free waters not required for L3 of several trichostrongyloid specieso migrate from the ground onto herbage, but is neededn order for L3 to leave sheep faeces. In contrast to larvalevelopment, however, no studies have yet attempted touantify rainfall requirements for L3 migration out of fae-es after the worm eggs had hatched and the larvae hadeveloped to L3. The aims of the work presented here areo determine the extent to which free water is necessary

or migration of H. contortus L3 out of faecal pellets, andow this is influenced by the hydration status of faeces andhe ambient environmental conditions. To a limited extenturvival of larvae in desiccated dung was also examined

logy 204 (2014) 258–264 259

in order to determine the relevance of sheep faeces as areservoir of L3 during drought.

2. Materials and methods

2.1. Conditions, equipment, faeces preparation and larvalrecovery during trials

All experiments were carried out in the laboratoryin order to avoid unpredictable confounding factors inthe field. Temperature and relative humidity (RH) wererecorded three times a day using an electronic hygrome-ter (FB70042 Traceable Hygrometer, Fisher Scientific, andU.K.), with temperature being maintained at 25 to 27 ◦C.Although the experiments were conducted in a room withno windows or external walls, RH appeared to be affectedto some degree by the weather, and varied from 17 to52%. Monoculture of H. contortus samples were providedby Moredun Research Institute (Edinburgh, U.K.). Basically,donor lambs were born and reared indoors to ensure thatthey were ‘worm free’. They were orally given 5000 H. con-tortus L3 in a tap water suspension delivered in a 5–10 mlsyringe. Twenty-one days post infection their egg countswere checked and the donor animal was harnessed if therewere sufficient eggs presented. Then their faeces were col-lected over a 24 h period with a traced bag. Infective larvaewere obtained by culturing faeces at 20 ◦C for seven days,in covered plastic containers (Versatile Environmental TestChamber, Sanyo, Japan). Air exchange was obtained byremoving the lids of the plastic containers of the culturesfor 2 min daily. All the faecal samples were well-formedpellets with a moist surface, and about 70% faecal moisturecontent (FMC).

The core element of the series of experiments was useof sieves 10 cm in diameter and of aperture 0.9 mm, onwhich faecal pellets were placed and subjected to varioussimulated rainfall treatments. The sieves were placed infunnels, in a modification of the standard Baermann appa-ratus and the funnels were mounted on wooden frames. Arubber hose with a clip at its loose end was connected tothe stem of each funnel in order to collect sediment con-taining infective larvae. The funnels were half filled withwater, with the sieve suspended above the water surface.Twenty-four hours after artificial rainfall had been applied,larval sediment was collected in test tubes by loosening theclip on the rubber hose beneath the funnels. This first col-lection consisted of L3 that were washed from the faecesby the simulated rain. Faecal pellets were then carefullytransferred to a new, dry sieve by tweezers, the old sievewas removed and larvae retained on the old sieve werethen recovered by placing it in a second funnel and raisingthe water level to submerge the sieve. Larval sediment wasthen recovered 24 h later. While the first funnel containedany L3 which had managed to migrate spontaneously fromthe pellets or to the pellet surface before the applicationof rain, the second collection represented larvae that hadactively migrated out of the faeces, during the course of theexperiment.

Each larval recovery comprised collection of the sedi-ment in 10 ml of water in a test tube. The number of larvaerecovered was estimated by inverting the tube severaltimes to thoroughly to bring the sediment into suspension,

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Parasitology 204 (2014) 258–264

Table 1Experiment 1: Effect of simulated rainfall on the migration of Haemonchuscontortus L3 from sheep faeces in a dry environment.

Day Simulated rainfall

Heavy Light

1–3 No rain No rain

indoor environment without any additional RH controlswhile the high RH Group was again, as above, placed inlarge sealed plastic boxes containing a shallow layer ofwater. Four replicates were monitored in each group. A

Table 2Experiment 2: Effect of relative humidity on the movement of Haemonchuscontortus L3 in the absence of simulated rainfall.

Day Relative humidity

260 T. Wang et al. / Veterinary

and transferring a 1 ml aliquot to a nematode countingcell (Sedgewick-Rafter Cell S50, PYSER-SGI, UK) with apipette. L3 were counted under ×40 total magnification.To adjust for differences in faecal moisture content (FMC)between different groups, the number of L3 recovered wererecorded as larvae per gram of dry matter (lpgDM). Todetermine FMC, subsamples of faeces were weighed beforeand after drying in an oven for 10 h at 80 ◦C.

Rainfall was simulated using a pressurised gardensprayer (Spraymist 1.25l, Hozelock, UK), with settings cal-ibrated to produce 2 mm of rainfall over the area of eachsieve over 3 s. The amount of rainfall delivered was checkedregularly by spraying over cylindrical beakers and measur-ing the water volume after fixed spraying times.

2.2. Statistical approach

Statistical methods are described for each experimentbelow, and conducted using PASW v18.0 software (SPSSInc., USA). The approach used analysis of variance (ANOVA),with increasing complexity according to the study design:thus, t-tests for comparing two groups, one-way ANOVA forcomparing a single variable across more than two groups,and General Linear Modelling (GLM) for comparing a vari-able across groups that differed in more than one factor.Proportions were arcsine-transformed before analysis inorder to stabilise the variance.

2.3. Experiment 1: Effect of rainfall on larval migrationfrom faeces in a dry environment

The first experiment aimed to confirm that rain wasnecessary for larval emergence from faeces, and furtherto investigate whether the amount of rainfall would affectthe extent of L3 migration from sheep dung in a dry envi-ronment. Sheep faeces containing H. contortus eggs werecultured to the L3 stage and 3 g of pellets were depositedon sieves, which were placed in funnels as described above.Two treatment groups were included in the trial: HeavyRain and Light Rain, each of which contained four repli-cates. The experiment lasted for 8 days: no rainfall wasapplied in the first three days. Thereafter, for the HeavyRain Group, 20 mm of simulated rain was applied withthe sprinkler on the fourth day, in the form of 10 smallequal treatments (2 mm) spread at 40-min intervals over7 h, whereupon no more simulated rain was given for theHeavy Rain Group after the fourth day. In the Light RainGroup, small rainfall treatments of 2 mm were simulatedonce daily from day 4 to day 7. For both groups, larval sed-iment from each replicate was collected daily from eachof the funnels on which a sieve had been suspended asdescribed above. At the end of the experiment, faecal pel-lets were crushed by digital pressure and subjected to theBaermann method to recover any live L3 which had failed tomigrate out of the faeces (Table 1). The proportion of L3 that

were known to be present in the faeces at the start of theexperiment (=the sum of those recovered from all sedimentcollections plus the final Baermann recovery) was recordedat each time step, and compared between treatments

4 10 × 2 mm 1 × 2 mm5–7 No rain 1 × 2 mm daily8 Baermannisation

by t-test. Based on the results of the first experiment, threefurther experiments were designed.

2.4. Experiment 2: Effect of RH on L3 movement in theabsence of rain

The second experiment considered the effect ofRH on movement of L3 from faecal pellets in theabsence of rain. Four sealed plastic food storage boxes(23.5 cm × 16.5 cm × 10 cm) were used to simulate veryhumid (saturated) and moderately humid environments.Two boxes were set up per treatment, each containingtwo replicates. In the moderate humidity treatment, RHwas maintained between 55 and 60% by placing a smallcontainer of saturated calcium nitrate solution in the boxalongside the samples. In the high RH group, a smallamount of water was placed in the bottom of the boxesto maintain RH at 98%. RH was monitored twice daily usinghygrometers as described above. Sieves and hygrometerswere held above the water or salt solution. FMC was deter-mined once daily by weighing one faecal pellet before andafter drying in an oven at 80 ◦C for 2 h. No rainfall wasapplied until day 9, when a large rainfall treatment (20 mmover 7 h) was applied to all replicates. Larvae migratingout of the faeces were counted as described above. Allthe faeces were subjected to the Baermann method onday 10 to recover L3 remaining in the pellets (Table 2).The proportion of L3 recovered from the two groups wasarcsine-transformed and compared using the t-test, withtreatment as a factor.

2.5. Experiment 3: Effects of both RH and rainfall on L3migration

This experiment investigated the influence of free wateron larval migration from sheep dung in both low and highRH conditions. The low RH Group was placed in the dry

Moderate (55–60%) High (up to 98%)

1–8 No rain No rain9 20 mm 20 mm10 Baermannisation

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Table 3Experiment 3: Effect of both relative humidity and simulated rainfall onL3 migration of Haemonchus contortus L3 from sheep faeces.

Relative humidity

Low High

Locality Room Sealed boxRelative humidity 31–52% 94–98%Day 1–3 No rain

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Fig. 1. Recovery of Haemonchus contortus L3 (mean ± standard errors)from sheep faeces subjected to heavy rain (20 mm on day 4, over a periodof 7 h, indicated by the solid arrow) and light rain (2 mm per day from days4 to 7, indicated by the dotted arrows). The remaining larvae were recov-ered from the samples by the Baermann method on day 8. Total meannumber of larvae recovered after rain in each treatment (range betweenreplicates) was 2829 (649) in the heavy rain group and 67 (54) in the lightrain group. The proportion of L3 emerging refers to the proportion of all

Day 4–8 2 mm rain per dayDay 9 Baermannisation

mall amount (2 mm) of rainfall was administered daily toll replicates from day 4 to day 8. FMC was measured beforeainfall events every day, and all the replicates were Baer-annised on day 9 (Table 3). The number of L3 recoveredas converted to three formats: (i) number of L3 recov-

red each day as a proportion of the total number lateround to have been present; (ii) number of L3 recovered as

proportion of those remaining in faeces on each day; (iii)umulative proportion of total larvae present that emergedach day during the 8-day experiment. All the proportionsere arcsine transformed, and analysed in a general lin-

ar model (GLM) that included two levels of RH (low andigh) and five collection time points (day 4 to day 8), withvaluation of the interaction between RH and days.

.6. Experiment 4: Effect of different amounts of rainfalln L3 migration

In order to quantify how much rain is needed for lar-al migration, a further study was carried out using theame experimental set up in a room environment wherehe average RH was 41%. Different amounts of daily rainfall4 mm, 6 mm and 8 mm delivered in 2 mm aliquots every

h) were applied from day 2 to day 7 and two replicatesere included in each treatment (Table 4). The proportions

f L3 recovered under these three rainfall regimes wereompared using one-way ANOVA, with rainfall amount asactor. Post-hoc Tukey tests were applied to test for differ-nces between treatments.

. Results

The results of each experiment are presented in turn.hroughout, an average of 70% (standard deviation 17%) ofarvae were found in the second collection (on the sieve)fter rainfall application rather than from the first, indicat-

ng that most larvae left the faeces by active migration. Forubsequent analyses, numbers of larvae recovered by eachethod are pooled.

able 4xperiment 4: Effect of different amounts of rainfall on the migration ofaemonchus contortus L3 from sheep faeces (relative humidity 41%).

Group Daily rainfall (from day 2 to 7) (mm)

1 2 × 2 mm2 3 × 2 mm3 4 × 2 mm

those recovered during the migration experiment plus by Baermann’s atthe end of the experiment.

3.1. Experiment 1: Effect of rainfall on larval migrationfrom faeces in a dry environment

Results of this experiment are shown in Fig. 1. In theHeavy Rain group, no L3 emerged from faecal samples inthe absence of simulated rainfall over the first three days.By 24 h after application of Heavy Rain (20 mm over 7 h),an average of 44% of L3 were recovered outside the faeces.This decreased sharply to 7% in the next 24 h, when no fur-ther rain was applied, after which a negligible number oflarvae emerged. By contrast, in the Light Rain group, veryfew larvae emerged at any stage. The arcsin-transformedproportion of larvae recovered one day after rainfall wassignificantly greater in the heavy than the light rain group(t = 4.006, p = 0.011). A large proportion of total L3 wasrecovered in both groups by the Baermann method on thefinal day (71% for Light Rain and 46% for Heavy Rain).

3.2. Experiment 2: Effect of RH on L3 movement fromfaecal pellets

Daily FMC and larval emergence in the two groups(moderate and high RH) throughout the duration of theexperiment are shown in Fig. 2.

One-way ANOVA on the total proportion of L3 thatemerged showed a significant difference in total larvalrecovery between moderate RH and high RH environ-ments when no rainfall was applied (F1,6 = 16.11, p < 0.05),with higher proportions of larvae recovered from the highRH group (mean proportion recovered = 0.057, S.D. = 0.027)than the moderate RH group (mean = 0.0027, S.D. = 0.0032).Although a significant difference was found between thesetwo groups statistically, the overall proportion of larvaerecovered was still very low (<10%) compared with thatrecovered after rainfall. Following rainfall treatment on day

9, an appreciable proportion of larvae (mean of 0.159 undermoderate and 0.247 under high RH) was recovered. Themean proportion of total larvae recovered by Baermann

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Fig. 2. (A) Daily faecal moisture content (FMC) in high relative humidity(98% RH) and moderate RH (55–60% RH) groups; (B) Proportion of totalL3 present that emerged over the experiment (±standard errors) in mod-erate and high RH groups before (light shaded bars, left) and after (darkshaded bars, right) heavy rainfall treatment. Total mean number of larvaerecovered in each rain treatment (range between replicates) was 2440(785) in the moderate RH group and 2602 (937) in the high RH group. A

Fig. 3. L3 larval emergence (mean ± standard errors) over time expressedin different formats. (A) L3 recovered daily as a proportion of the totalnumber of L3 eventually recovered; (B) L3 recovered daily as a proportionof the number remaining in faecal pellets on that day; (C) Cumulativeproportion of total larvae present that emerged each day. A small amount(2 mm) of rainfall was administered daily to all replicates from day 4 to

4. Discussion

total of 20 mm of rain was applied on day 9.

treatment after the nine day experiment was 0.84 in themoderate RH group and 0.68 in the high RH group.

3.3. Experiment 3: Effects of both RH and rainfall on L3migration

RH varied from 31 to 52% in the moderate humiditygroup, and 94 to 98% in the high humidity group. FMCin the high RH group was maintained at around 70–80%,while in the low RH treatment, faeces dried rapidly overthe first two days and then remained at FMC around 10%.Virtually no larvae were recovered from either group dur-ing the first three days, without rainfall treatment. Under2 mm of daily rainfall from day 4, significant numbers ofL3 were recovered in the high RH group, followed by adecrease from day 5, as faecal pellets became denudedof L3. In contrast, the larvae emerging from the low RHgroup were much more limited in number. Fig. 3 showseffects of the two treatments on L3 recovery rate. Itappears that in the high RH treatment, approximately 40%of remaining larvae migrated from the faeces per daywhen 2 mm rain were applied, whereas in the low RHtreatment, the proportion of larvae migrating was con-siderably lower. The GLM showed that when applying2 mm daily rainfall treatment, L3 recovery rate expressedas a proportion of larvae remaining in the faeces waspositively influenced by RH (p < 0.001). There was also

a significant interaction between collection day and RH(p < 0.05).

day 8. Total mean number of larvae recovered in each treatment (rangebetween replicates) was 2726 (531) in low RH group and 5215 (1795) inhigh RH group.

3.4. Experiment 4: Effect of different amounts of rainfallon L3 migration

There was no significant difference between differentrainfall treatments on the total proportion of L3 recovered(F2,27 = 0.49, p = 0.952) (Fig. 4). In the low RH environmentof all three trial groups, arithmetic mean recovery of L3indicated that the biggest release of L3 took place after thefirst rain day, with as much as 49% of total L3 recoveredfrom faeces in 4 mm group, 54% in the 6 mm group and38% in the 8 mm group. Success of L3 migration was notinfluenced by the amount of rainfall across the range tested.

The effect of simulated rainfall on larval emergencefrom sheep faeces was tested under a range of conditions.

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Fig. 4. Proportion of Haemonchus contortus L3 (mean ± standard errors)emerging from sheep faeces each day under different rates of artifi-cial rainfall, in dry conditions. L3 are expressed as a proportion of thetotal number eventually recovered. Rain was administered from day 2 todbt

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ay 7. Total mean number of larvaere covered in each treatment (rangeetween replicates) was 5175 (1344) in the 4 mm group, 8516 (187) inhe 6 mm group and 1021 (438) in the 8 mm group.

ree water in the form of simulated rainfall was neededor appreciable L3 migration out of faeces. The amount andemporal distribution of rainfall were found to be impor-ant to the number of free L3 recovered, as well as RH,uch that in dry conditions light rain was insufficient totimulate migration, whereas heavy rain yielded L3. Thisiffered in moist conditions, in which faecal moisture con-ent (FMC) was maintained by light, regular rainfall and L3ere then able to emerge. Provided that FMC and rainfallere sufficient to support migration, progressive increases

n rainfall amount did not significantly increase the numberf L3 recovered. At ambient temperatures between 25 and7 ◦C, and conditions of low regular rainfall on moist faeces,pproximately 40% of the remaining L3 left the faeces eachay. It is worth noting that faeces used in this study wereegular pellets. Softer, moister faeces can agglomerate intoarger masses, potentially affecting the rate of migrationnd the total proportion of larvae that are able to leave theaeces.

The study demonstrates the key role that FMC plays in3 migration from faecal pellets, in conjunction with rain-all. FMC is an integrated variable determined by the ratiof cumulative precipitation and evaporation (P/E), RH in thenvironment, and temperature on the faecal surface. Thus,MC combines a number of important climatic factors thatonsiderably influence the development and migration ofnfective larvae. The importance of FMC could hinge onhe ability of a partly hydrated faecal surface and/or inter-al matrix to support larval motion. This is supported byhe finding that 2 mm of rainfall was not enough to allow

igration in dry conditions, while both higher rainfall origher RH with low rainfall were able to support migra-ion, presumably by restoring suitable conditions for larval

ovement within the faecal pellets. Rossanigo and Gruner1995) reported that 70% FMC provided optimal conditions

or the development of eggs of H. contortus. Gronvold andoghschmidt (1989) reported low Ostertagia ostertagi lar-al movement from cow pats after simulated rainfall onaeces with a dry surface, but high translation with even

logy 204 (2014) 258–264 263

splash droplets on faeces with a moist surface. It might benecessary to explore the ‘splash theory’ on sheep faecesthough differences clearly exist between the discrete pelletof sheep and larger faecal mass of cattle.

In the high RH (high FMC) groups of this study, it is pos-sible that even a small amount of rainfall could soften thefaecal pellet surfaces sufficiently for L3 migration becauseof moisture transmission within the pellets. The results ofExperiment 1 show this pattern clearly; after heavy rain-fall was applied on day 4, a significant number of L3 wasrecovered on day 5, and this then decreased sharply to neg-ligible levels within 48 h. This might be attributed to theextremely low RH in the environment (mean 25%), whichmust have resulted in rapid drying of the faecal pellets afterfree water treatment on day 4, thus constricting movementof the L3 within them.

Recently, the importance of soil moisture on the devel-opment of GIN has been highlighted (Khadijah et al., 2013a)and is likely to act, alongside other climatic and environ-mental factors, through FMC. Soil moisture could affectmovement of L3 out of faeces in a similar way. Translationof L3 onto pasture in the absence of rainfall under condi-tions of high soil moisture has been described (Khadijahet al., 2013b), and could be explained by the availabilityof free water in the soil matrix and/or surface under suchconditions, facilitating larval migration.

The present study also gave some indication of the abil-ity of H. contortus L3 to tolerate desiccation in dry faeces,even though over much shorter periods of time than inthe trials of others (Berbigier et al., 1990; Rossanigo andGruner, 1995; Van Dijk and Morgan, 2011). In the absenceof rainfall, as in Experiment 2, no L3 migrated from thelow FMC (moderate RH) group during the eight-day exper-iment. However, many viable L3 were still recovered byBaermannisation on the last day. Different genera of GINvary in their ability to tolerate desiccation. For instance,both eggs and pre-infective L1 and L2 of Trichostrongyluscolubriformis are more resistant to desiccation than thoseof H. contortus (Berbigier et al., 1990; Rossanigo and Gruner,1995). However, good survival of trichostrongylid L3 insheep faeces has been previously demonstrated (Van Dijkand Morgan, 2011), and sheep faeces therefore have thepotential to act as reservoirs of L3 in a similar way tothose of cattle (Stromberg, 1997). However, longer dura-tion experiments and field studies would be needed todetermine the epidemiological significance of such sur-vival.

The attempt in Experiment 4 to estimate the effect ofabove-threshold rainfall on the rate of L3 migration showedno significant difference in L3 recovery between faecesreceiving 4, 6 and 8 mm of daily rainfall. Given that 2 mmof daily rainfall in this environment failed to release sub-stantial numbers of L3, a minimum of between 2 and 4 mmof daily rainfall seems to be needed for larvae to leave thefaeces, with additional rainfall having no further effect. Thethreshold for migration is likely to vary with FMC and hencewith RH and other factors, and could be higher than this in

drier environments.

It has been generally believed that the epidemiologyof H. contortus depends significantly on the short termmicroclimatic situation rather than long term variation in

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climate, thus the level of L3 on pasture might be alteredwithin a few days. Such phenomena are most typical insemi-arid areas, where large numbers of parasites can beseen after rainfall following numerous dry months thathave prevented parasite transmission (Agyei, 1997). Inorder to move from faeces onto pasture, L3 must firstleave the dung, and then move horizontally and verticallyonto herbage. Since free water is not necessary for verti-cal movement of larvae on grass, at least under conditionsof moderate RH (Van Dijk and Morgan, 2011), the positivecorrelation between L3 recovery and free water must arisemostly from effects on the release of larvae from faecalpellets. While L3 can survive for long periods in desic-cated faeces, there can be practically no migration of L3from such faeces in the absence of rainfall above a certainthreshold required to soften the crust and/or matrix. Fur-thermore the latter can be expected to vary according tothe physical state of the faeces. This study has not estab-lished with certainty the threshold levels involved, but diddemonstrate that both FMC and rainfall amount are crucialfactors determining the migration of H. contortus L3 fromfaecal pellets.

Further studies are needed to more fully investigateeffects of climate on FMC, to guide FMC manipulation inlaboratory experiments, and to understand effects of rain-fall on larval migration at pasture. Given the apparentimportance of soil moisture to FMC and larval availability(Khadijah et al., 2013a,b), the impact of top soil moistureand the vegetation mat on larval movement from faeces toherbage deserve further experimental attention.

Acknowledgements

This work received funding from BBSRC–DFID CIDLIDproject BB/H00940X/1 RISCNET and from EU projectFP7-KBBE-2011-5-288975 GLOWORM. We thank twoanonymous reviewers for their constructive comments.

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