MIGRATION OF INFECTIVE LARVAE OF SHEEP GASTROINTESTINAL NEMATODES

The misration ofinfective larvae of nematodes of the genffl8 HaertIOIIdrJu, Ostmagia, Trichostrongylus and CIrabm/Q on berbale in the soil was studied under laboratory conditions on three artificially seeded srass plots with different methods of irription. The capacity for vertical migration was found to be the hishest, i. e. up to 27 em, in infective larvae of Trichostrongylus spp. Most of the infective larvae of this nematode were found in the berbqe 6-15 em above the soil surface. The rate of vertical migration is proportional to the moisture of the berbage. Durina 3 bours of watering the berbage, infective larvae were found 12-15 em above the soil surface, wbereas ,within 4 bours of dessication of the berbage the larvae were concentrated maximally up to 4 em from the soil surface. Infective larvae of H. contortus, TrichostrongylUs spp. and Chabertia ovintl misrated borizontally to a maximal distance of 21 em from the middle of the faeces sample establisbed on srass irripted twice a day. Infective larvae of the above nematode species occurred only sporadically in soil in the depth of 2 em; only in one case were infective larvae of Ostertagia spp. found in a depth of 7 em.

in infective larvae of Trichostrongylus spp.Most of the infective larvae of this nematode were found in the berbqe 6-15 em above the soil surface.The rate of vertical migration is proportional to the moisture of the berbage.Durina 3 bours of watering the berbage, infective larvae were found 12-15 em above the soil surface, wbereas ,within 4 bours of dessication of the berbage the larvae were concentrated maximally up to 4 em from the soil surface.
Infective larvae of H. contortus, TrichostrongylUs spp.and Chabertia ovintl misrated borizontally to a maximal distance of 21 em from the middle of the faeces sample establisbed on srass irripted twice a day.
Infective larvae of the above nematode species occurred only sporadically in soil in the depth of 2 em; only in one case were infective larvae of Ostertagia spp.
found in a depth of 7 em.l'lfective larvae, herbage, soil, rate qfmlgration From the epizootiological point of view, the infective larvae occurring in the berbage of pastures are one of the most important sources of distnbution of belminth invasions.
Several authors have studied the annual occurrence of infective larvae in the external environment, especially on herbage.B ii r g e r et al. (1983) reported that larvae began to migrate from the faeces to the berbage at the time wben the average daily air temperature bad exceeded 18 °C for more than a week and after at least 4 days of rainfall when average precipitation was minimally 20 mm.Can ina n and W est cot t (1986) stated that the occurrenCe of larvae on herbage was significantly dependent on the temperature and humidity of the environment ' In the berbage of the studied pastures (in November) in South Australia, 0 v ere n d et al.
( 1984) found as much as 4800 infective larvae per 1 kg of dry grass.Pro s I (1986) studied the quantity of larvae on vegetation during the day and found that the coUilts of infective O. oster-tQgi larvae were maximum at 6.00 a. in.0. e. 24701kg of fresh grass) and minimum in the course of the day 0. e. 3901kg offresb grass).,

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In general, in exPeriments ,conducted directly on pasturellind, infective larvae misrate horizon-, tally to a distance of 10-12 em, and vertically 17 em.Fin c be r and S t e war t (1979) reported that O. ostertag/larvae in the field migrated vertically at least 12.5 em, and in glass tubes in the laboratory 15 em (from the soil).On the contrary, C a II ina n and We s tcot t (1986) found 71% of the larvae recovered from the vegetation concentrated, WitbinO-2 em of the herbage.P r o!ll (1986) stated that larvae misrated vertically on theberbage only a few em and that during day they descended downwards to the lower grass layer above the soil.
Del gad 0 (1983) recovered infective Trichostrongylus and Haemonchus larvae on grass 50-130 em above the ground R 0 s e (1961) found the infective larvae to migrate more vertically than horizontally.He found the counts of larvae to be the bighest in tufts of grass near the faeces.Go I•d b erg (1970) collected 80% of O. ostertagi, C punct(lta and O. radlalUm larvae on grass within 13 em of the faeces.Pro s 1 (1986) found infective Ostertagia and Cooperia larvae on herbage as much as 40-50 em from the faeces, the majority of larvae being collected within 10 em of the faeces. .In addition to the migration of larvae. in herbage, many authors also investigated the migration oflarvae into the soil in connection with their sUrvival during the cold periods and their higher occurrence on herbage during spring and in the dry period.Calion a n and We s t cot t (19116) recovered 8 t~ more larvae in the soil than on the herbage down to a depth of 4-6 em.A 1 S a qua r et al. (1982) collected soil samples from October till September and found that infective Ostertagia spp.larvae were present .ineach sample to a depth of at least 15 em.Analysis of the vertical distribution suggested a possible migration into the soil during winter followed by an upward trend during spring and summer.G run e r et al. (1982) observed that towards the end of winter, during two weeks when soil temRerature was lower than 6°C, the larvae migrated to a depth of 40 em, and more.This migration through the soil was supported by the content of soil water, but the larvae were capable of active migration upwards even under relatively dry conditions.R 0 s e und Sma 11 (1985) stated that the ability of larvae to migrate into the soil was not even affected by the period of heavy rainfall.The results of studies of these and other authors have proved that infective larvae of many nematodes migrate horizontally over the herbage .muchmore quickly than in the opposite direction -into the soil.These authors concluded that the soil does not serve as a reservoir where large numbers of infective larvae might survive during the winter.
The mechanism of larvae migration in the soil has not yet been fully explained.Based on his experiment with O. ostertagi larvae, G ron vol d (1979) suggested that in this respect the earthworms might play a certain role; after 50 days he isolated living larvae from their digestive tract and faeces.
. The aim of the present study was to fmd the ability and rate of migration of infective larvae of the most frequently occurring sheep gastrointestinal namatodes on artificially established grassland, and to discuss the presence of infective larvae in soil as a possible reservoir of infective larvae under unfavourable conditions.

MaterbdslDdMethods
Various ways of migration on infective nematode larvae of the genera Haemonchus, Ostertagia, Trichostrongylus and Chobertitl were studied.Positive faeces with eggs of the above helminths were collected from spontaneously infected sheep and placed on the experimental plots (see further) under laboratory conditions.

(A) Characteristics of the experimental plots
The vertical and horizontal migration of infective larvae was investigated in March and April in a greenhouse on three artificially seeded experimental plots (35 X 56 em).The composition of the herbage was as follows: Festuca pratensis, Trifolium repens, Loiium peTe1Ine and Dactylis glomerata.(B) Methods of examinations Within 35 d&ys of establishment of the faeces samples with the eggs of the above nematode species, the herbage and soil of the experimental plots were divided into squares 7 X 7 em.
The high herbage was gradually cut into 3 em parts, from the soil surface up to the top.
Soil samples were taken to a depth of 2 and 7 em.In pilot field samplings, the soil was taken on the experimental plots in the Ecological Station Klec in April and November to a depth of 20 em from 5 X 5 em squares.
All samples were examined for infective nematode Iarvae of the genera Haemonchus, OsterttJgIa, Trichostrongylus and Chabertia using the modifiechnetbod according to Bearmann.
(C) Determination_of the rate of migration of the infective larvae The rate of migration of the infective Iarvae in the herbage was ascertained in three 13 X 13 em containers into which the grass mats were set, and the faeces with the infective larvae were over the whole surface.The herbage was continuously watered.
Samples of the herbage were taken gradually from these 8 plots at 15 and 30 min intervals, then at 1 to 6-hour intervals.The gnI$S was cut in 2 em parts and samples were examined using the modified method according to Baermann.The ability of reverse migration was investigated after dessication using warm air (45°C).

I. Vertical and horizontal migration
The average air temperature and relative humidity during the whole experimental period from the establishment of the experiment till its termination was 21.3 DC (from 12 DC to 28 DC) and 74% (from 52% to 85%), respectively.
(1) Migration of infective Iarvae on the herbage The results are.given in Fig. 1 and 2. Fig. 1 shows that on the regularly watered plot the infective Iarvae migrated horizontally only in the middle part of the experimental plot, maximally 21 em from the middle of the faeces sample (part A).At this distance, infective larvae of Trichostrongylus spp.Chabertia ovilUl and Haemonchus contOrtus were recovered.Within 14 em of the centre of the plot, infective Iarvae of Trichostrongylus spp.and CkovilUl were collected.
As concerns vertical migration of larvae, the fJgUres in the middle of the squares of Fig. 1 give the maximal height (in em) from the soil surface where infective larvae were recovered on the herbage.The present investigations proved that infective larvae of Trichostrongylus spp.had the highest ability of migration.i.e. up to a height of 27 em.Infective H. contOrtus, Ostertogia spp.and CkovilUl larvae were found up to a height of 6 em, 15 em and 9 em, respectively, from the faeces.In cut herbage (part B). infective Iarvae of Trichostro1lR)llus spp.Ostertagia spp.and H. contOrtus were recovered.Fig. 2 shows the migration of infective larvae on herbage on an unwatered plot with bottom watering.No horizontal migration of infective Iarvae was observed on this experimental plot and vertical migration occurred only on the high herbage in three squares above 'the faeces.Examinations of grass samples from these squares showed only the occurrence of infective Iarvae of Trichostrongylus spp. up to a height ofJ8 em.
On the third experimental plot (watered, with regularly distributed faeces), the maximal ability for vertical migration of infective larvae was examined.Infective larvae of Trichostrongylus spp.wert found to migrate most frequently up to a maximal height of 27 em, the ~ority being collected 6-15 em above the soil sur/oce.Infective larvae of Ostertogia spp.were recovered from grass samples of 3-9 em in height Infective larvae of H. contOrtus were found in two samples only, i.e. 27-30 em and 15-18 em above the soil. (

n. Rate of migration
No infective Jarvae were found on herbage within 15,30 and 60 minutes at l? °C and relative air humidity 4O'MI.After 2 hou.rs, infective Jarvae of HMmonchus cont~, Trichostrongylils, spp., ~ Bpp. and ChIIbertia ovina were ~vered on the herbqe' up, to' 8 em above the soil; after 3 hours, larvae were found 12 -IS em above the soil.' At 22 C C and 45 -50% relative air humidity, infective larvae were found on herbage 2 em, 6 em. and 12-15 em above the soil within 30 minutes, 1 hour, 2 hours and 3 hours, respectively.The infective larvae of all the species, i. e. Ostertagia spp., Trichostrongylus SPP.,Haemonchus contonus and Chabertia avina, were found to migrate.
Reverse migration was also investigated; the herbage with the infective larvae was dessicated with warm air (the temperature above the herbage was 25 °C and relative air humidity 30%) and within 1 hour, 2 hours and 4 hours of dessication infective larvae were found in a height of 10 em, 6 em and 4 em, respectively.Results of investigations of infective larvae miJration in h~ on experlineiiiat plot N o . 2 " .''(', , '.'

Discussion
From our observations it is apparent that infective larvae of the genera Hoemonchus.Trichostrongylus.Ostmllgia and Chabntla migrated vertically.a lesser proportion horizontally.Infective larvae were collected on herbage only above the faeces or in the close vicinity.R 0 s e (1961) reported that infective larvae migrated mor~ vertically than horizontally and that the largest counts of larvae were recovered from.grass tufts near the faeces in the lower layer of the herbage.In their studies, G 0 I d b erg (1970) and Pro s I (1986) described horizontal misration.
In our experiment, the infective larvae of Trichostrongylus spp., at most, migrated vertically. up to a height of 27 em; the rruVority of Haemonchus.Trichostrongylus.OsterttJgla and Chabertia larvae found at a height of 6-15 em above the soil with faeces.The results of Fin c her and S t e war t (1979) and Del gad 0 (1983) were similar.On the contrary, C a II ina n and W est cot t (1986) and Pro s I (1986) collected infective larvae m the lower layers of the herbage.
If the infective larvae are to use their ability for migration, they require humidity which is maintained for a longer time in the dense and high herbage.This fact agrees with the present results when infective larvae on the experimental plot regularly watered, migrated up to a height of 27 -30 em, while they remained in the faeces on the plot with bottom watering.
From the present results it further follows that the misration of infective larvae into the soil is probably not markedly affected either by the permeability or moisture of the soil.Infective larvae of H. contortus.Ostertagia spp.and Trichost1"OligyIitt••spp;,: were recovered from the soil only sporadically, and only to a depth of 2 em; only-ia::Otte;~e Ostertagia spp.larvae were recovered in a depth of 7 em.It is true that in orientltiql1:rreld samplings carried out in November, infective larvae were found to reach a depth,oq() ,~but this does not conclusively prove the ability of infPive larvae to migrate into th~ SOu,:1)puse we cannot exclude contamination during sampliDg or if the larvae were carried ~~bt.Percolating water during rainfall.The present results agree with results of B 0 r g s t ee d. '8Ild•B 0 0 gar d (1983), R 0 s e and Sma II (1985) who recovered very small amountf:Of ).m:ective larvae in the soil.On the other hand, A I Sag u r et al. (1982), G rune .tt:~'•al> (1982) and C a II ina n and W est cot t (1986) reported that infective larvae IIliJi'ided deeply into the soil.The mechanism of larvae migration.inthe soil has not yet been ,fully explained; as mentioned above, some authors attach importance to concrete local conditions, others suggest the role of earthworms in the transport of infective larvae in the dift"erent'layers and depths of the soil.
Observations on the rate of migration have revealed that, under sufficient air humidity and adequate temperature, the infective Haemonchus.Osterfagitl, Trichostrongylus an,d Charb£rtia larvae are capable of reaching a height of 15 em on the herbage within 3 hours.During dessication, the infective larvae migrate downwards again and remain at a height of up to 4 em where humidity is sufficient The onset of migration of the larvae in the herbage is dependent on the temperature and humidity of the environment that is in accordance with the observations of B ii r g e r et al. (1983) and C: a II ina n and W est cot t (1986).
The ability of infective larvae to migrate quicldy in the herbage, including their ability of surviving the relatively verY unfavourable environment81 conditions, is a very serious epizootiological problem.K 0 t r I a and K 0 f d 0 i\ (1978)' observed that not eve~ recurrent winter temperatures .•connected with hoarfrost decontaminated the pastures.According to these authors, 53% of the herbage samples on the expeQmental plot were• positive from June till June of the following year.Based on observations of 0 v ere ,n d et al. (1984) and P 0 s I (1986), the pastures are infested with considerable numbers of• infective larvae during the whole grazing season.In their studies, Cab are t et al. (1982), Mit c h e I and Fit z s i m 0 n s (1983) and Mit c h e I et al. (1984) concentrated their attention to testing various types of pastures.
They forind that sYstems of so-called "clean pastules" (i.e.past\lres which had Dot been.grazed for 12 months, or pastures ploughed and sown in the autumn and not grazed until the follOwing autumn) did partly reduce the hazard of infesting the grazing animals, especially the young ones, ~ut after a certain period these pastures were infested.
From the present results and from data of other authors quoted in this study, it is apparent that also at the present time the pasture herbage is the main source of distribution of helminthia-.ses; pastures remain to be a very serious epizootiological problem in spite of the fact that the present methods of, tfe8ting' pastures have• QUalitatiwly improVed'u compared witil'tf1e' paSt' (a) Experimental plot No. 1 Positive.faeces were placed on the grass surface into the centre of the plot in the form of a cake 21 em in diameter.Before placing the faeces, one half of the plot of grassland was kept at a height of 2 em frequent clipping.The herbage on the other half of the plot was 50 em high before the beginning of the experiment The herbage was watered ~cea~. .(b) Experimental plot No.2 The herbage and establishment of faeces were the same as on plot No.1.Only once a day, the bottom 20 em of soil were irrigated by immersing into a large pot with water for 15 minutes.(c) Experimental plot No.3 On the third experimental plot the faeces were evenly distributed over the whole surface.The herbage was not cut and was irrigated ~ce a day as was experimental plot No.1; at the time of collection of larvae it reached a height of 50 em.
Fig. 1Results of investiptions of infective Jarvae misration in herbqe on experimental plot No. 1