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Evolution of nematode community in grazing sheep selected for resistance and susceptibility to Teladorsagia circumcincta and Trichostrongylus colubriformis: a 4-year experiment
Veterinary Parasitology 109 (2002) 277–291
Evolution of nematode community in grazing sheep selected for resistance and susceptibility to Teladorsagia circumcincta and Trichostrongylus colubriformis: a 4-year experiment L. Gruner a,∗ , J. Cortet a , C. Sauvé a , C. Limouzin b , J.C. Brunel c a b
INRA, Bio-Agresseurs, Santé et Environnement, 37380 Nouzilly, France INRA, UE Pathologie Aviaire et Parasitologie, 37380 Nouzilly, France c INRA, Domaine de La Sapinière, 18390 Osmoy, France
Received 25 April 2002; received in revised form 13 August 2002; accepted 23 August 2002
Abstract Field trials were undertaken to compare nematode population dynamics in two experimental flocks of rams selected for high and for low faecal egg counts (FECs) following two doses with 20,000 infective larvae, 12,000 Teladorsagia circumcincta (Tcirc) and 8000 Trichostrongylus colubriformis (Tcol) separated by a treatment. The selection was done by dosing 200 ram lambs (INRA 401 breed) and FECs proceeded with egg identification on days 28 and 35 post-each-infection. The 30 lambs with the lowest FECs constituted the group of permanent resistant rams (R), the 30 with the highest values the group of susceptible ones (S). Each group grazed separated pastures from April to November, this during 4 consecutive years. The contamination (number of eggs deposited) was estimated every 3 weeks on each pasture. The larval populations was measured by successive groups of tracer lambs. The infection of the permanent rams was done by slaughtering eight R and S rams on mid and end of each grazing season. Each autumn, new R and S ram lambs were selected to complete the R and S groups before turning out in spring. On third year, a third species (Haemonchus contortus, Hcon) was added to compare its behaviour with the two others. The regulation of parasite populations were studied by comparing what happens in R rams and pasture to S ones. The selected R ram lambs had a FEC of Tcirc 50% lower than S ones. This induced a similar lowered pasture contamination, a 25% decrease of infective larval population in years 2 and 3, and the worm burdens in R rams were the half of that in S rams after 2 years. The FEC of Tcol was 75% lower at the selection and induced similar difference in pasture contamination, but 65 and 96% reduction in L3 population, respectively, after 1 and 2 years. The worm burden was reduced by 99%. Adding Hcon species during the third year, in year 4, the L3 population ∗ Corresponding author. Tel.: +33-2-4742-7751; fax: +33-2-4742-7774. E-mail address: [email protected] (L. Gruner).
0304-4017/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 0 1 7 ( 0 2 ) 0 0 3 0 2 - 3
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was half in R pasture compared with S one, and R rams had 60% less worms of this species than S ones. This selection without any drench prevent outbreaks observed in young tracer lambs on S pasture. The comparison of selected resistant rams to selected susceptible ones and not to unselected controls gave more rapid information about the tendency of the regulation of the communities of nematode parasites. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Sheep-nematoda; Teladorsagia circumcincta; Trichostrongylus colubriformis; Haemonchus contortus; Population dynamics; Genetics; Resistance
1. Introduction Breeding for resistance to gastrointestinal nematode parasites appeared as a possible way to contribute to the control of strongylosis. Albers and Gray (1986) demonstrated that the egg output can be considered indirectly to measure worm burden and is of interest as it also measures the contamination of pasture. The heritability of egg counts was estimated in different breeding and environmental situations (Morris, 1998). This demonstrates that this trait could be included in a breeding scheme. The economic interest of breeding for resistant animals is difficult to estimate and Woolaston and Baker (1996) discussed about the choice of the best objective. One objective can be a reduction of the egg output, to reduce pasture contamination and the level of infection of the flock. Bisset et al. (1997) studied this kind of selection in Romney sheep under New Zealand conditions. Another aspect concerns the ability of the hosts to produce when parasitised, called resilience. In New Zealand, Bisset et al. (1994) and Morris et al. (1995) studied the feasibility of selecting for reduced treatments. In a tropical situation where Haemonchus contortus (Hcon) is the dominant species, the heritability of the faecal egg output was estimated in different sheep and goat breeds and presented low values in some resistant local breeds such as the hair sheep Red Maasai (Baker et al., 1998). In the West Indies, Mandonnet et al. (2001) found significant heritability in 10-month-old kids, but in this kind of humid tropics, we do not know if the selection for lowering the contamination of pastures is of interest and this needs to be investigated. Under temperate climate, significant heritability estimates were found in situations where Teladorsagia circumcincta (Tcirc) was the dominant species in Blackface sheep in Scotland (Bishop et al., 1996), and in Polish long wool sheep in south Poland where T. circumcincta and H. contortus were the main species of the nematode communities (Bouix et al., 1998). But until now, this trait was not introduced in a selection scheme. The aim of the experiment as reported herein was to demonstrate if sheep selected based on low faecal egg counts (FECs), resulted in lowered pasture contamination and lead to control of worm infections in the flock. To do this, a resistant and a susceptible group of 30 ram lambs were selected from 200 rams after two doses with a mixture of T. circumcincta and Trichostrongylus colubriformis (Tcol) larvae and bred during 4 consecutive years on separated pastures. The number of eggs deposited on the pastures, of infective larvae and of worms in the flocks were regularly estimated.
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2. Materials and methods 2.1. Selection of resistant and susceptible animals The sheep breed INRA 401, a fixed cross-breed Romanov–Berrichon du Cher was developed at the INRA experimental farm “La Sapinière”, as being prolific and having a good lamb conformation for meat production. Two hundred ram lambs born in September 1995 from 175 ewes mated with 30 rams were kept in penned conditions. Transported to INRA Nouzilly, they received when 4 months old two doses with a mixture comprising 12,000 L3 of Tcirc and 8000 L3 of Tcol. The total of 20,000 L3 corresponded to the doses of Tcirc used in previous experiments to estimate the genetic parameters of resistance of Romanov sheep to this species (Gruner et al., 1998). As the fecundity of Tcirc female worms is less important than in Tcol, each dose comprised more L3 of this species. The two doses were separated by a drenching (fenbendazole) at day 33 after the first one, and 2 weeks removal; individual FECs were done days 26 and 33 after each infection. The 200 lambs were ranked with the mean of the four FECs (after square root transformation) and two groups of 30 lambs were selected, a resistant (R) and a susceptible (S) one, respectively, with the lowest and the highest FEC values. This regimen to rank all the lambs was chosen from the cited previous experiments (Gruner et al., 1998), the resistance of each individual being better defined with more than one infection and more than a single FEC after each infection. Each year a portion of the rams were necropsied, so new selections were processed in “La Sapinière” experimental farm to replace them. As lambs were grazing during the autumn, FECs were processed in November and if the level of infection was too low (this occurred in 1997), a dose of 10,000 Tcirc L3 was given and additional FECs done. The natural infection in this area was predominantly Tcirc (more than 80% of the L3 after faecal culture). 2.2. Experimental design and data collection 2.2.1. Preparation of the two pastures Two separate newly seeded pastures (1.65 ha each, rye grass and white clover) without any parasite were grazed 10 days in early April 1996 with two groups of 60 lambs (remaining after the selection of R and S groups), equilibrated on their weight and FEC. This permitted the deposition in each pasture of about 140 million eggs of Tcirc and 250 million eggs of Tcol. 2.2.2. Experimental design The R and S groups of rams were grazing from mid-April to mid-November and housed during the winter, from 1996 to 1999. FECs and weight were done every 3 weeks. At the end of June and end of pasture season, eight rams from each group (five from the first year) were kept 3 weeks in penned conditions before their necropsy for worm burden estimation. New R and S ram lambs selected during the autumn were introduced before turning out to complete each group to 30 permanent rams. As a consequence, in year 1 all rams were 1 year-old; in year 2, half were 1 year-old and half 2 year-old; in year 3 as on year 4, half were 1 year-old, 1/4 2 year-old and 1/4 3 year-old. The slaughtered rams had similar proportions of categories of age (Fig. 1).
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Fig. 1. Experimental scheme of permanent resistant (R) or susceptible (S) rams and tracer lambs (tr) grazing on pasture R and S, and penned before slaughtering (sla) or housed during winter time, this during each year from 1996 to 1999.
The level of larval infection of the pastures was estimated each grazing season by the use of five groups of six 3–4-month-old tracer lambs in each pasture, grazing 18 days (to prevent any egg deposal). They were penned 3 weeks before their necropsy. To investigate what happens if a third species was introduced, in July of the third year, the two tracer groups were previously infected with 1500 L3 of Hcon, this to deposit eggs from this new species during their 18-day grazing period. 2.2.3. Laboratory measures To facilitate the identification of the eggs of the two or three species during the FECs, faeces cooled at 4 ◦ C when collected, were kept 24 h at 10 ◦ C then 20 h at 12 ◦ C before the FEC. Tcirc eggs were at embryonic state, those of Tcol at tadpole state, and those of Hcon did not initiate their development. Their size and morphology also help their identification. The contamination of the pastures was the number of eggs of each nematode species deposited by the permanent rams during successive 3-week periods, estimated as number of eggs per gram (epg) multiplicated by the faeces excretion. A constant value of 41 g of fresh faeces/kg of metabolic live weight was used, measured in penned conditions for this sheep breed. 2.2.4. Climatic conditions Temperature and rainfall were recorded at the INRA experimental farm less than 500 m from the two pastures. The total rainfall during each grazing seasons was 388, 472, 604 and 727 mm, respectively, from 1996 to 1999 (Fig. 2).
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Fig. 2. Monthly rainfall and temperatures during the four grazing seasons at the experimental farm of INRA, Nouzilly (France).
3. Results 3.1. Constitution of R and S groups of rams The 200 ram lambs were ranked on their mean epg and R and S groups of 30 lambs selected with the lowest and the highest epg values. The distribution of these R and S groups for each nematode species (Fig. 3) showed that the two groups were totally separated for Tcol with 5-fold more eggs excreted by S compared with R lambs. For Tcirc, there are some overlapping between the two groups, S lambs excreting in average 3.4-fold more eggs than R ones. 3.2. Contamination of the pastures R and S Each flock grazed its own pasture and eggs were only deposited by R and S permanent rams, the tracer lambs leaving the pasture before excreting any egg. An exception concerned the first deposal of Hcon eggs in July 1998. The number of deposited eggs of each species (Fig. 4) presented annual and seasonal variations related to climatic conditions. On the two pastures, each year the contamination with Tcirc eggs increased during the spring. It was higher in year 2 compared to the first one, and, in years 3 (1998) and 4 (1999), quickly
Fig. 3. Distribution of the 30 resistant and 30 susceptible lambs within the 200, selected on their mean FECs of T. circumcincta and T. colubriformis.
Fig. 4. Number of millions of eggs from each gastrointestinal nematode species deposited by the rams on the pastures R and S during the four grazing seasons.
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Fig. 5. Mean T. circumcincta, T. colubriformis and H. contortus worm burdens in tracer lambs grazing the pastures R and S.
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decreased after the first few weeks the rams were turned out. Pasture S received about twice as many eggs than the R one. This difference was greater for the contamination with Tcol, being nearly nil in R pasture, and with Hcon. 3.3. Larval infection of the pastures R and S The successive five groups of tracer lambs gave a good estimation of the populations of infective larvae of Tcirc and Tcol present on the pastures during the grazing season of each year (Fig. 5). The first year, the deposition in early April of 140 million Tcirc eggs and 250 million Tcol eggs, completed by favourable climatic conditions with some hot weeks, permitted an unexpected and unusual important larval infection on the two pastures, nearly similar between pastures R and S. Later on, pasture S was more infected with Tcirc larvae than pasture R, reflecting differences of contamination. Tcol populations were low in the second year, nearly nil on pasture R. This species only reappeared the last autumn on this pasture. The larval populations of Hcon remained at a very low level in autumn 1998 and moderate level after summer 1999. 3.4. Infection of the R and S rams The worm burdens of the rams necropsied in mid and late grazing season (Table 1) presented significant seasonal and annual variations with higher values in autumn compared with the end of spring, out of the peculiar first year. They were generally lower in R rams compared with the S ones, especially for Tcol species, but also for Hcon species. In November 1999, more Tcol worms were observed in R rams, and more than 75% of the worms of Hcon were at the early L4 stage. 3.5. Regulation of worm population by R and S rams A way to estimate the regulation done by the permanent rams is to compare their worm burdens with the cumulated number of worms in the tracer lambs grazing during the same period Table 1 Worm burdens (back transformed geometric means) of the eight R and S rams necropsied in mid and end of each grazing season on their pasture R or S Grazing period 23 April–24 June 1996a 23 April–14 November 1996a 7 April–17 June 1997 7 April–4 November 1997 14 April–12 June 1998 14 April–3 November 1998 15 April–15 June 1999 15 April–2 November 1999 a b
T. circumcincta
T. colubriformis
H. contortus
Rams S
Rams R
Rams S
Rams R
Rams S
Rams S
5,394 3,120 4,425 5,106 14,239 27,000 12,888 4,796
7034 1094 1939 2933 2959 5670 2168 2907
8511 103 63 7881 147 872 382 2969
119 3 3 219 2 40 7 15 252
9 209 14 612b
5 10 5745b
Only five rams necropsied. About 70–85% as E L4 only in H. contortus.
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of time (Fig. 6). From each pasture, rams necropsied at the end of June and mid-November ingested more than the cumulated number of worms found in tracer lambs (after adjustment of the duration of grazing). For the 4 years we have eight pairs of data per group. If we defined the resistance of the rams as their worm burden expressed as the percentage of the cumulated number of worms in the tracer lambs (Fig. 6), it is more clear that when few worms were in tracers, 100% remained in the permanent rams. When more than 10,000 Tcirc worms were ingested and established, a rapid regulation occurred, and the percentage decreased. This regulation was a little more efficient in R rams: less than 20% of the ingested worms remained present when the cumulated number of worms in the tracer lambs reached 15–20,000. In S rams, this level of regulation is observed after a cumulated number of 30–40,000 worms. The regulation of Tcol population is similar in S rams but is very early initiated in R rams. No sufficient data were available for Hcon to do this comparison. 3.6. Comparison of nematode regulation between R and S flocks Another way to estimate the regulation of the worm populations for each nematode species is to compare what happens in R flock and R pasture relatively to S ones (Table 2). In the initial selection, R ram lambs had an egg output of Tcirc 50% lower than in S ram lambs. During the total duration of the grazing season, the contamination of the pasture R was the half of that of pasture S. The larval population (measured in tracer lambs) was diminished by 25% every year and the worm population in the flock of R rams was the half of that in S rams after 2 years, a quarter after 3 years. For Tcol species, the effect was more efficient. R ram lambs were selected with an egg output 75% lower than in S ram lambs; this induced a similar decrease in the contamination during the total duration of the grazing period. Larval population was lowered by 65% in year 1, 99% in year 2 and worm burdens lowered to 1% after 1 year. The ram lambs were not selected against Hcon but the contamination was lowered by 80%, the larval population by 50 and the worm burden by 60% in 1 year. 3.7. Effects on production traits The body weight of the rams increased during the 4 years as the flocks only comprised 1-year-old weaned lambs at the beginning of the first grazing season, then older animals in Table 2 Balance sheet of the evolution of the populations of the three nematode species in a flock R initially selected with low FECs of T. colubriformis and T. circumcincta, referred to a flock S initially selected on their high FECs of the two same species Trait
T. circumcincta
T. colubriformis
H. contortus
Lower initial selection on FECs in R referred to S flock
50% lower
75% lower
Not determined
50% 25% in 1 year 50% in 2 years, 75% in 3 years
75% 65% in 1 year, 96% in 2 years 99% in 1 year
80% 50% in 1 year 60% in 1 year
Consequence on The pasture contamination (%) The larval population on pasture The worm burden in rams
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the other years. Differences between R and S rams (data not shown) were more significant during autumn of the 2 last years (more humid with higher levels of infection). It was decided to drench with fenbendazole treatment individuals having negative weight-gain during two consecutive weighings and FECs above 3000 epg. This only occurred in eight S rams the first year (four in June, one in September and three in October). Tracer lambs spent a too short time on pastures to be able to present valuable modifications in their weight-gain. But the last groups of lambs in 1998 and in 1999 on S pasture collected so high quantity of infective larvae of Tcir and Tcol that the majority of them died or were quickly necropsied some days after leaving the pasture. In average, they got 34,000 Tcirc plus 33,000 Tcol worms on 3 November 1998, 9000 and 36,000 on 2 November 1999. On R pasture, only three lambs died on 3 November 1999 with about 8000 and 21,000 worms of Tcirc and Tcol, respectively. 4. Discussion 4.1. The experimental flocks Our limited experimental possibilities restricted the size of the two flocks to 30 sheep, so it was decided to keep only rams and not ewes with their lambs. So our results mainly concerned ram schools and not common commercial flocks, even if the use of tracer lambs permitted to get information in young animals in complement with those got in adult animals. Only resistant and susceptible groups of sheep were compared and an unselected control one was not present. However this permitted to determine the tendency of what would happen by breeding sheep selected with low FECs, at a speeder scale than by comparing with an unselected group. The identification of the eggs at the species level was possible since only two or three known species were concerned. This permitted to compare the regulation of the two coexisting populations of Tcirc and Tcol and observe what happens with the introduction of Hcon species. No similar experiment was published; Bisset et al. (1997) did a 2-year experiment with lambs issued from two divergent breeding lines and separately bred on two farms. 4.2. Epizootiological observations during the running 4-year experiment The initial selection of the resistant and susceptible groups of ram lambs was efficient as the R group excreted 5-fold less Tcol eggs and 3-fold less Tcirc eggs. The replacement ram lambs were selected in natural conditions or after a supplemental dose, as Gruner et al. (1998) demonstrated that the response to natural and experimental infection had a genetic correlation of 0.98. The main modification was the fact that natural selection mainly concerned Tcirc as very few Tcol existed in this situation. Between the 4 years of the experiment, the first year was peculiar due to the unusual high level of contamination of the R and S pastures with Tcirc and Tcol eggs, early in the season. The other years were more representative of the epidemiology of gastrointestinal nematode parasites in this part of France with low levels of infective larvae in the early spring, predominantly of Tcirc species, Tcol being more abundant after the summer, and
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Tcirc being predominant again in late autumn (Gruner et al., 1980). From year 2 and more evidently in years 3 and 4, the contamination with Tcirc eggs increased in early spring but suddenly decreased at the end of May (Fig. 4), even with high levels of L3 on grass as seen in tracer lambs (Fig. 5). Many FECs had nil values, in R as in S rams. This can be explained by the rapid turnover of the Tcirc population, the presence of L3 populations inducing a self-cure. This corresponded to the shape of Tcirc egg output in groups of lambs grazing clean pastures but receiving from 500 to 5000 L3 per day (Coop et al., 1985); after a maximum 4 weeks after the beginning of infection, FECs declined to very low values in week 6 then increased again in weeks 10–14. As a consequence, worm counts are not a good reflection of egg output all year long, but remain the best indicator of resistance. Luffau et al. (1981) used the self-cure to estimate the resistance of sheep to infection and re-infection with Hcon. An important difference was observed in the populations of Tcol, more numerous in S pasture, in tracers as in S rams necropsied in mid and end of first grazing season. This species nearly disappeared in R pasture until the last autumn where it was quite abundant in tracer lambs. R rams necropsied in November 1999 had higher burdens than S rams. The explanation could be that S rams were always in contact with this species, stimulating their resistance. Inversely, the R rams were nearly without any contact with Tcol L3 since mid 1996, so they highly react to this new Tcol population and they were necropsied before having the time to built an efficient resistance. 4.3. Worm burdens in permanent rams compared with burdens in tracer lambs By comparing the worm burdens in rams with the cumulated burdens of tracer lambs grazing the same period of time, Anderson (1972) illustrated when grazing sheep acquired resistance during the season. In his work, 23 groups of four lambs grazing 2 weeks and six groups of four adults were slaughtered during the year. Due to the high diversity of individual response, this work is more descriptive of the evolution of the 1-year study than quantitative. The aim of our study was to get more quantitative data. So we increased the number of tracer lambs to six per group and the number of rams to eight per group to get more valuable means. Each of the five groups of tracers grazed 18 days totalling 90 days distributed during the total grazing time of the rams (197–210 days). This permitted to have a good estimate of the evolution of the infection during each grazing season. We necropsied permanent rams at only two critical times not to make a kinetic but a balance sheet of what happens during the half or the total duration of the grazing season. We obtained two values per year for R and S rams for the two main parasite species, representing eight situations differing with seasons, year and pasture infection history (Fig. 6). So by expressing the worm burdens in adults as a percentage of the cumulated number of worms in tracer lambs, a huge regulation was evidenced over 10,000 Tcirc worms ingested and established in tracer lambs. It was a little more efficient in R than in S rams (where it occurred over about 20,000 worms). For Tcol, this regulation was initiated over 10,000 worms in S rams but very early in R rams. The too low number of larvae of this species did not permit a correct estimate of this regulation. The point of R rams necropsied in November 1999 was out of the others due to the huge and recent infection met by this group.
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4.4. Comparison between R and S groups Bisset et al. (1997) compared ewes–lambs issued from resistant line to counterparts issued from susceptible line of Romney sheep. At the end of the two experimental grazing seasons, pasture with resistant lambs harboured 5–6-fold less infective larvae, the egg output being more than 50-fold lower. Infection was natural and consisted of a mixture of nematode species. By comparing our experiment, we demonstrated the huge difference existing in the regulation of the two main nematode species Tcirc and Tcol. For Tcirc, with a pasture contamination divided by 2, the larval intake decreased by 25% per year, the worm burden by 50% in 2 years, 75% in 3 years. The difference was higher with Tcol, from the selection step to the worm burden which decreased by 99% in 1 year, compared with susceptible rams. Even if replacement rams were mainly selected upon their resistance status with Tcirc, R rams were more resistant to Tcol. Results obtained during the first year were probably in excess due to the unusual high level of spring contamination; more average level would delay the acquired resistance. We added H. contortus during the summer of year 3 (this species existed in this part of France and occurred at the end of the summer). The larval population decreased by 50% in 1 year, the worm burden by 60%, demonstrating that selection for resistance to Tcol gave a good control to Hcon. Windon (1990) also found that the selected line high responder to Tcol vaccination had a good heterologous control when challenged with other species including Hcon. Our experiment was not built to have a correct estimate of the economical interest of the breeding sheep selected for low FECs, but some observations are of interest. In year 1, all the rams were 1 year old, so we found more individuals with more than 3000 epg and/or losing weight during two successive periods of 3 weeks than in the other years. These individuals were dewormed. Only one treatment was done the other grazing seasons, resulting in an increase of strongylosis in S pasture and rams. S rams were suffering the two last summers of the high level of infection. The most important effect was observed in the tracer lambs; all died after grazing only 18 days the two last autumns on S pasture. Selection to limit the pasture contamination permitted to prevent this outbreak in year 3. Inversely, selection so highly limited the population of Tcol during 3 years on R pasture that the absence of antigenic stimulation did not prevent half of the tracer lambs to die with 20,000 worms of Tcol nor R rams to collect 15,000 worms. This too extreme situation is possibly abnormal, but a new population can be rebuilt from a low level of larval infection of the pasture and low egg output in 1 or 2 months with favourable climatic conditions difficult to forecast. Our experiment was conducted with a limited number of species, and this limited diversity in nematode community is in accordance with theories and examples demonstrating that stability and limited high levels of infections are more related with high diversity (Silvestre et al., 2000). 5. Conclusions A selection on reducing T. circumcincta egg output permitted to limit the infection with this species with a lower efficiency compared with the selection on low T. colubriformis egg output. In this case, the species nearly disappeared, preventing outbreaks observed on
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pasture with susceptible rams, but a new population can be rebuilt in some months at a dangerous level. Selection on resistance to this species also gave a good protection to H. contortus.
Acknowledgements We thank J. Bouix from the INRA “Amélioration Génétique des Animaux” unit, for his valuable discussions necessary to elaborate the experiment. We are grateful to the technical assistance of the team of the experimental farm ‘La Sapiniere’ where the INRA 401 sheep were produced and selected. We are also grateful to J. Auger, T. Chaumeil, and P. Vincent for their technical participation for the management of the experiment at INRA Nouzilly.
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Morris, C.A., Watson, T.G., Bisset, S.A., Vlassoff, A., Douch, P.G.C., 1995. Breeding sheep in New Zealand for resistance or resilience to nematode parasites. In: Gray, G.D., Woolaston, R.R., Eaton, B.T. (Eds.), Breeding for Resistance to Infectious Disease in Small Ruminants. Monograph Series No. 34. Australian Centre for International Agricultural Research (ACIAR), Canberra, pp. 259–274. Silvestre, A., Chartier, C., Sauvé, C., Cabaret, J., 2000. Relationship between helminth species diversity of infection and breeding management in dairy goats. Vet. Parasitol. 94, 91–105. Windon, R.G., 1990. Selective breeding for the control of nematodiasis in sheep. Rev. Sci. Tech. Off. Int. Epiz. 9, 555–576. Woolaston, R.R., Baker, R.L., 1996. Prospects of breeding small ruminants for resistance to internal parasites. Int. J. Parasitol. 26, 845–855.
Evolution of nematode community in grazing sheep selected for resistance and susceptibility to Teladorsagia circumcincta and Trichostrongylus colubriformis: a 4-year experiment L. Gruner a,∗ , J. Cortet a , C. Sauvé a , C. Limouzin b , J.C. Brunel c a b
INRA, Bio-Agresseurs, Santé et Environnement, 37380 Nouzilly, France INRA, UE Pathologie Aviaire et Parasitologie, 37380 Nouzilly, France c INRA, Domaine de La Sapinière, 18390 Osmoy, France
Received 25 April 2002; received in revised form 13 August 2002; accepted 23 August 2002
Abstract Field trials were undertaken to compare nematode population dynamics in two experimental flocks of rams selected for high and for low faecal egg counts (FECs) following two doses with 20,000 infective larvae, 12,000 Teladorsagia circumcincta (Tcirc) and 8000 Trichostrongylus colubriformis (Tcol) separated by a treatment. The selection was done by dosing 200 ram lambs (INRA 401 breed) and FECs proceeded with egg identification on days 28 and 35 post-each-infection. The 30 lambs with the lowest FECs constituted the group of permanent resistant rams (R), the 30 with the highest values the group of susceptible ones (S). Each group grazed separated pastures from April to November, this during 4 consecutive years. The contamination (number of eggs deposited) was estimated every 3 weeks on each pasture. The larval populations was measured by successive groups of tracer lambs. The infection of the permanent rams was done by slaughtering eight R and S rams on mid and end of each grazing season. Each autumn, new R and S ram lambs were selected to complete the R and S groups before turning out in spring. On third year, a third species (Haemonchus contortus, Hcon) was added to compare its behaviour with the two others. The regulation of parasite populations were studied by comparing what happens in R rams and pasture to S ones. The selected R ram lambs had a FEC of Tcirc 50% lower than S ones. This induced a similar lowered pasture contamination, a 25% decrease of infective larval population in years 2 and 3, and the worm burdens in R rams were the half of that in S rams after 2 years. The FEC of Tcol was 75% lower at the selection and induced similar difference in pasture contamination, but 65 and 96% reduction in L3 population, respectively, after 1 and 2 years. The worm burden was reduced by 99%. Adding Hcon species during the third year, in year 4, the L3 population ∗ Corresponding author. Tel.: +33-2-4742-7751; fax: +33-2-4742-7774. E-mail address: [email protected] (L. Gruner).
0304-4017/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 0 1 7 ( 0 2 ) 0 0 3 0 2 - 3
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was half in R pasture compared with S one, and R rams had 60% less worms of this species than S ones. This selection without any drench prevent outbreaks observed in young tracer lambs on S pasture. The comparison of selected resistant rams to selected susceptible ones and not to unselected controls gave more rapid information about the tendency of the regulation of the communities of nematode parasites. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Sheep-nematoda; Teladorsagia circumcincta; Trichostrongylus colubriformis; Haemonchus contortus; Population dynamics; Genetics; Resistance
1. Introduction Breeding for resistance to gastrointestinal nematode parasites appeared as a possible way to contribute to the control of strongylosis. Albers and Gray (1986) demonstrated that the egg output can be considered indirectly to measure worm burden and is of interest as it also measures the contamination of pasture. The heritability of egg counts was estimated in different breeding and environmental situations (Morris, 1998). This demonstrates that this trait could be included in a breeding scheme. The economic interest of breeding for resistant animals is difficult to estimate and Woolaston and Baker (1996) discussed about the choice of the best objective. One objective can be a reduction of the egg output, to reduce pasture contamination and the level of infection of the flock. Bisset et al. (1997) studied this kind of selection in Romney sheep under New Zealand conditions. Another aspect concerns the ability of the hosts to produce when parasitised, called resilience. In New Zealand, Bisset et al. (1994) and Morris et al. (1995) studied the feasibility of selecting for reduced treatments. In a tropical situation where Haemonchus contortus (Hcon) is the dominant species, the heritability of the faecal egg output was estimated in different sheep and goat breeds and presented low values in some resistant local breeds such as the hair sheep Red Maasai (Baker et al., 1998). In the West Indies, Mandonnet et al. (2001) found significant heritability in 10-month-old kids, but in this kind of humid tropics, we do not know if the selection for lowering the contamination of pastures is of interest and this needs to be investigated. Under temperate climate, significant heritability estimates were found in situations where Teladorsagia circumcincta (Tcirc) was the dominant species in Blackface sheep in Scotland (Bishop et al., 1996), and in Polish long wool sheep in south Poland where T. circumcincta and H. contortus were the main species of the nematode communities (Bouix et al., 1998). But until now, this trait was not introduced in a selection scheme. The aim of the experiment as reported herein was to demonstrate if sheep selected based on low faecal egg counts (FECs), resulted in lowered pasture contamination and lead to control of worm infections in the flock. To do this, a resistant and a susceptible group of 30 ram lambs were selected from 200 rams after two doses with a mixture of T. circumcincta and Trichostrongylus colubriformis (Tcol) larvae and bred during 4 consecutive years on separated pastures. The number of eggs deposited on the pastures, of infective larvae and of worms in the flocks were regularly estimated.
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2. Materials and methods 2.1. Selection of resistant and susceptible animals The sheep breed INRA 401, a fixed cross-breed Romanov–Berrichon du Cher was developed at the INRA experimental farm “La Sapinière”, as being prolific and having a good lamb conformation for meat production. Two hundred ram lambs born in September 1995 from 175 ewes mated with 30 rams were kept in penned conditions. Transported to INRA Nouzilly, they received when 4 months old two doses with a mixture comprising 12,000 L3 of Tcirc and 8000 L3 of Tcol. The total of 20,000 L3 corresponded to the doses of Tcirc used in previous experiments to estimate the genetic parameters of resistance of Romanov sheep to this species (Gruner et al., 1998). As the fecundity of Tcirc female worms is less important than in Tcol, each dose comprised more L3 of this species. The two doses were separated by a drenching (fenbendazole) at day 33 after the first one, and 2 weeks removal; individual FECs were done days 26 and 33 after each infection. The 200 lambs were ranked with the mean of the four FECs (after square root transformation) and two groups of 30 lambs were selected, a resistant (R) and a susceptible (S) one, respectively, with the lowest and the highest FEC values. This regimen to rank all the lambs was chosen from the cited previous experiments (Gruner et al., 1998), the resistance of each individual being better defined with more than one infection and more than a single FEC after each infection. Each year a portion of the rams were necropsied, so new selections were processed in “La Sapinière” experimental farm to replace them. As lambs were grazing during the autumn, FECs were processed in November and if the level of infection was too low (this occurred in 1997), a dose of 10,000 Tcirc L3 was given and additional FECs done. The natural infection in this area was predominantly Tcirc (more than 80% of the L3 after faecal culture). 2.2. Experimental design and data collection 2.2.1. Preparation of the two pastures Two separate newly seeded pastures (1.65 ha each, rye grass and white clover) without any parasite were grazed 10 days in early April 1996 with two groups of 60 lambs (remaining after the selection of R and S groups), equilibrated on their weight and FEC. This permitted the deposition in each pasture of about 140 million eggs of Tcirc and 250 million eggs of Tcol. 2.2.2. Experimental design The R and S groups of rams were grazing from mid-April to mid-November and housed during the winter, from 1996 to 1999. FECs and weight were done every 3 weeks. At the end of June and end of pasture season, eight rams from each group (five from the first year) were kept 3 weeks in penned conditions before their necropsy for worm burden estimation. New R and S ram lambs selected during the autumn were introduced before turning out to complete each group to 30 permanent rams. As a consequence, in year 1 all rams were 1 year-old; in year 2, half were 1 year-old and half 2 year-old; in year 3 as on year 4, half were 1 year-old, 1/4 2 year-old and 1/4 3 year-old. The slaughtered rams had similar proportions of categories of age (Fig. 1).
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Fig. 1. Experimental scheme of permanent resistant (R) or susceptible (S) rams and tracer lambs (tr) grazing on pasture R and S, and penned before slaughtering (sla) or housed during winter time, this during each year from 1996 to 1999.
The level of larval infection of the pastures was estimated each grazing season by the use of five groups of six 3–4-month-old tracer lambs in each pasture, grazing 18 days (to prevent any egg deposal). They were penned 3 weeks before their necropsy. To investigate what happens if a third species was introduced, in July of the third year, the two tracer groups were previously infected with 1500 L3 of Hcon, this to deposit eggs from this new species during their 18-day grazing period. 2.2.3. Laboratory measures To facilitate the identification of the eggs of the two or three species during the FECs, faeces cooled at 4 ◦ C when collected, were kept 24 h at 10 ◦ C then 20 h at 12 ◦ C before the FEC. Tcirc eggs were at embryonic state, those of Tcol at tadpole state, and those of Hcon did not initiate their development. Their size and morphology also help their identification. The contamination of the pastures was the number of eggs of each nematode species deposited by the permanent rams during successive 3-week periods, estimated as number of eggs per gram (epg) multiplicated by the faeces excretion. A constant value of 41 g of fresh faeces/kg of metabolic live weight was used, measured in penned conditions for this sheep breed. 2.2.4. Climatic conditions Temperature and rainfall were recorded at the INRA experimental farm less than 500 m from the two pastures. The total rainfall during each grazing seasons was 388, 472, 604 and 727 mm, respectively, from 1996 to 1999 (Fig. 2).
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Fig. 2. Monthly rainfall and temperatures during the four grazing seasons at the experimental farm of INRA, Nouzilly (France).
3. Results 3.1. Constitution of R and S groups of rams The 200 ram lambs were ranked on their mean epg and R and S groups of 30 lambs selected with the lowest and the highest epg values. The distribution of these R and S groups for each nematode species (Fig. 3) showed that the two groups were totally separated for Tcol with 5-fold more eggs excreted by S compared with R lambs. For Tcirc, there are some overlapping between the two groups, S lambs excreting in average 3.4-fold more eggs than R ones. 3.2. Contamination of the pastures R and S Each flock grazed its own pasture and eggs were only deposited by R and S permanent rams, the tracer lambs leaving the pasture before excreting any egg. An exception concerned the first deposal of Hcon eggs in July 1998. The number of deposited eggs of each species (Fig. 4) presented annual and seasonal variations related to climatic conditions. On the two pastures, each year the contamination with Tcirc eggs increased during the spring. It was higher in year 2 compared to the first one, and, in years 3 (1998) and 4 (1999), quickly
Fig. 3. Distribution of the 30 resistant and 30 susceptible lambs within the 200, selected on their mean FECs of T. circumcincta and T. colubriformis.
Fig. 4. Number of millions of eggs from each gastrointestinal nematode species deposited by the rams on the pastures R and S during the four grazing seasons.
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Fig. 5. Mean T. circumcincta, T. colubriformis and H. contortus worm burdens in tracer lambs grazing the pastures R and S.
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decreased after the first few weeks the rams were turned out. Pasture S received about twice as many eggs than the R one. This difference was greater for the contamination with Tcol, being nearly nil in R pasture, and with Hcon. 3.3. Larval infection of the pastures R and S The successive five groups of tracer lambs gave a good estimation of the populations of infective larvae of Tcirc and Tcol present on the pastures during the grazing season of each year (Fig. 5). The first year, the deposition in early April of 140 million Tcirc eggs and 250 million Tcol eggs, completed by favourable climatic conditions with some hot weeks, permitted an unexpected and unusual important larval infection on the two pastures, nearly similar between pastures R and S. Later on, pasture S was more infected with Tcirc larvae than pasture R, reflecting differences of contamination. Tcol populations were low in the second year, nearly nil on pasture R. This species only reappeared the last autumn on this pasture. The larval populations of Hcon remained at a very low level in autumn 1998 and moderate level after summer 1999. 3.4. Infection of the R and S rams The worm burdens of the rams necropsied in mid and late grazing season (Table 1) presented significant seasonal and annual variations with higher values in autumn compared with the end of spring, out of the peculiar first year. They were generally lower in R rams compared with the S ones, especially for Tcol species, but also for Hcon species. In November 1999, more Tcol worms were observed in R rams, and more than 75% of the worms of Hcon were at the early L4 stage. 3.5. Regulation of worm population by R and S rams A way to estimate the regulation done by the permanent rams is to compare their worm burdens with the cumulated number of worms in the tracer lambs grazing during the same period Table 1 Worm burdens (back transformed geometric means) of the eight R and S rams necropsied in mid and end of each grazing season on their pasture R or S Grazing period 23 April–24 June 1996a 23 April–14 November 1996a 7 April–17 June 1997 7 April–4 November 1997 14 April–12 June 1998 14 April–3 November 1998 15 April–15 June 1999 15 April–2 November 1999 a b
T. circumcincta
T. colubriformis
H. contortus
Rams S
Rams R
Rams S
Rams R
Rams S
Rams S
5,394 3,120 4,425 5,106 14,239 27,000 12,888 4,796
7034 1094 1939 2933 2959 5670 2168 2907
8511 103 63 7881 147 872 382 2969
119 3 3 219 2 40 7 15 252
9 209 14 612b
5 10 5745b
Only five rams necropsied. About 70–85% as E L4 only in H. contortus.
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of time (Fig. 6). From each pasture, rams necropsied at the end of June and mid-November ingested more than the cumulated number of worms found in tracer lambs (after adjustment of the duration of grazing). For the 4 years we have eight pairs of data per group. If we defined the resistance of the rams as their worm burden expressed as the percentage of the cumulated number of worms in the tracer lambs (Fig. 6), it is more clear that when few worms were in tracers, 100% remained in the permanent rams. When more than 10,000 Tcirc worms were ingested and established, a rapid regulation occurred, and the percentage decreased. This regulation was a little more efficient in R rams: less than 20% of the ingested worms remained present when the cumulated number of worms in the tracer lambs reached 15–20,000. In S rams, this level of regulation is observed after a cumulated number of 30–40,000 worms. The regulation of Tcol population is similar in S rams but is very early initiated in R rams. No sufficient data were available for Hcon to do this comparison. 3.6. Comparison of nematode regulation between R and S flocks Another way to estimate the regulation of the worm populations for each nematode species is to compare what happens in R flock and R pasture relatively to S ones (Table 2). In the initial selection, R ram lambs had an egg output of Tcirc 50% lower than in S ram lambs. During the total duration of the grazing season, the contamination of the pasture R was the half of that of pasture S. The larval population (measured in tracer lambs) was diminished by 25% every year and the worm population in the flock of R rams was the half of that in S rams after 2 years, a quarter after 3 years. For Tcol species, the effect was more efficient. R ram lambs were selected with an egg output 75% lower than in S ram lambs; this induced a similar decrease in the contamination during the total duration of the grazing period. Larval population was lowered by 65% in year 1, 99% in year 2 and worm burdens lowered to 1% after 1 year. The ram lambs were not selected against Hcon but the contamination was lowered by 80%, the larval population by 50 and the worm burden by 60% in 1 year. 3.7. Effects on production traits The body weight of the rams increased during the 4 years as the flocks only comprised 1-year-old weaned lambs at the beginning of the first grazing season, then older animals in Table 2 Balance sheet of the evolution of the populations of the three nematode species in a flock R initially selected with low FECs of T. colubriformis and T. circumcincta, referred to a flock S initially selected on their high FECs of the two same species Trait
T. circumcincta
T. colubriformis
H. contortus
Lower initial selection on FECs in R referred to S flock
50% lower
75% lower
Not determined
50% 25% in 1 year 50% in 2 years, 75% in 3 years
75% 65% in 1 year, 96% in 2 years 99% in 1 year
80% 50% in 1 year 60% in 1 year
Consequence on The pasture contamination (%) The larval population on pasture The worm burden in rams
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the other years. Differences between R and S rams (data not shown) were more significant during autumn of the 2 last years (more humid with higher levels of infection). It was decided to drench with fenbendazole treatment individuals having negative weight-gain during two consecutive weighings and FECs above 3000 epg. This only occurred in eight S rams the first year (four in June, one in September and three in October). Tracer lambs spent a too short time on pastures to be able to present valuable modifications in their weight-gain. But the last groups of lambs in 1998 and in 1999 on S pasture collected so high quantity of infective larvae of Tcir and Tcol that the majority of them died or were quickly necropsied some days after leaving the pasture. In average, they got 34,000 Tcirc plus 33,000 Tcol worms on 3 November 1998, 9000 and 36,000 on 2 November 1999. On R pasture, only three lambs died on 3 November 1999 with about 8000 and 21,000 worms of Tcirc and Tcol, respectively. 4. Discussion 4.1. The experimental flocks Our limited experimental possibilities restricted the size of the two flocks to 30 sheep, so it was decided to keep only rams and not ewes with their lambs. So our results mainly concerned ram schools and not common commercial flocks, even if the use of tracer lambs permitted to get information in young animals in complement with those got in adult animals. Only resistant and susceptible groups of sheep were compared and an unselected control one was not present. However this permitted to determine the tendency of what would happen by breeding sheep selected with low FECs, at a speeder scale than by comparing with an unselected group. The identification of the eggs at the species level was possible since only two or three known species were concerned. This permitted to compare the regulation of the two coexisting populations of Tcirc and Tcol and observe what happens with the introduction of Hcon species. No similar experiment was published; Bisset et al. (1997) did a 2-year experiment with lambs issued from two divergent breeding lines and separately bred on two farms. 4.2. Epizootiological observations during the running 4-year experiment The initial selection of the resistant and susceptible groups of ram lambs was efficient as the R group excreted 5-fold less Tcol eggs and 3-fold less Tcirc eggs. The replacement ram lambs were selected in natural conditions or after a supplemental dose, as Gruner et al. (1998) demonstrated that the response to natural and experimental infection had a genetic correlation of 0.98. The main modification was the fact that natural selection mainly concerned Tcirc as very few Tcol existed in this situation. Between the 4 years of the experiment, the first year was peculiar due to the unusual high level of contamination of the R and S pastures with Tcirc and Tcol eggs, early in the season. The other years were more representative of the epidemiology of gastrointestinal nematode parasites in this part of France with low levels of infective larvae in the early spring, predominantly of Tcirc species, Tcol being more abundant after the summer, and
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Tcirc being predominant again in late autumn (Gruner et al., 1980). From year 2 and more evidently in years 3 and 4, the contamination with Tcirc eggs increased in early spring but suddenly decreased at the end of May (Fig. 4), even with high levels of L3 on grass as seen in tracer lambs (Fig. 5). Many FECs had nil values, in R as in S rams. This can be explained by the rapid turnover of the Tcirc population, the presence of L3 populations inducing a self-cure. This corresponded to the shape of Tcirc egg output in groups of lambs grazing clean pastures but receiving from 500 to 5000 L3 per day (Coop et al., 1985); after a maximum 4 weeks after the beginning of infection, FECs declined to very low values in week 6 then increased again in weeks 10–14. As a consequence, worm counts are not a good reflection of egg output all year long, but remain the best indicator of resistance. Luffau et al. (1981) used the self-cure to estimate the resistance of sheep to infection and re-infection with Hcon. An important difference was observed in the populations of Tcol, more numerous in S pasture, in tracers as in S rams necropsied in mid and end of first grazing season. This species nearly disappeared in R pasture until the last autumn where it was quite abundant in tracer lambs. R rams necropsied in November 1999 had higher burdens than S rams. The explanation could be that S rams were always in contact with this species, stimulating their resistance. Inversely, the R rams were nearly without any contact with Tcol L3 since mid 1996, so they highly react to this new Tcol population and they were necropsied before having the time to built an efficient resistance. 4.3. Worm burdens in permanent rams compared with burdens in tracer lambs By comparing the worm burdens in rams with the cumulated burdens of tracer lambs grazing the same period of time, Anderson (1972) illustrated when grazing sheep acquired resistance during the season. In his work, 23 groups of four lambs grazing 2 weeks and six groups of four adults were slaughtered during the year. Due to the high diversity of individual response, this work is more descriptive of the evolution of the 1-year study than quantitative. The aim of our study was to get more quantitative data. So we increased the number of tracer lambs to six per group and the number of rams to eight per group to get more valuable means. Each of the five groups of tracers grazed 18 days totalling 90 days distributed during the total grazing time of the rams (197–210 days). This permitted to have a good estimate of the evolution of the infection during each grazing season. We necropsied permanent rams at only two critical times not to make a kinetic but a balance sheet of what happens during the half or the total duration of the grazing season. We obtained two values per year for R and S rams for the two main parasite species, representing eight situations differing with seasons, year and pasture infection history (Fig. 6). So by expressing the worm burdens in adults as a percentage of the cumulated number of worms in tracer lambs, a huge regulation was evidenced over 10,000 Tcirc worms ingested and established in tracer lambs. It was a little more efficient in R than in S rams (where it occurred over about 20,000 worms). For Tcol, this regulation was initiated over 10,000 worms in S rams but very early in R rams. The too low number of larvae of this species did not permit a correct estimate of this regulation. The point of R rams necropsied in November 1999 was out of the others due to the huge and recent infection met by this group.
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4.4. Comparison between R and S groups Bisset et al. (1997) compared ewes–lambs issued from resistant line to counterparts issued from susceptible line of Romney sheep. At the end of the two experimental grazing seasons, pasture with resistant lambs harboured 5–6-fold less infective larvae, the egg output being more than 50-fold lower. Infection was natural and consisted of a mixture of nematode species. By comparing our experiment, we demonstrated the huge difference existing in the regulation of the two main nematode species Tcirc and Tcol. For Tcirc, with a pasture contamination divided by 2, the larval intake decreased by 25% per year, the worm burden by 50% in 2 years, 75% in 3 years. The difference was higher with Tcol, from the selection step to the worm burden which decreased by 99% in 1 year, compared with susceptible rams. Even if replacement rams were mainly selected upon their resistance status with Tcirc, R rams were more resistant to Tcol. Results obtained during the first year were probably in excess due to the unusual high level of spring contamination; more average level would delay the acquired resistance. We added H. contortus during the summer of year 3 (this species existed in this part of France and occurred at the end of the summer). The larval population decreased by 50% in 1 year, the worm burden by 60%, demonstrating that selection for resistance to Tcol gave a good control to Hcon. Windon (1990) also found that the selected line high responder to Tcol vaccination had a good heterologous control when challenged with other species including Hcon. Our experiment was not built to have a correct estimate of the economical interest of the breeding sheep selected for low FECs, but some observations are of interest. In year 1, all the rams were 1 year old, so we found more individuals with more than 3000 epg and/or losing weight during two successive periods of 3 weeks than in the other years. These individuals were dewormed. Only one treatment was done the other grazing seasons, resulting in an increase of strongylosis in S pasture and rams. S rams were suffering the two last summers of the high level of infection. The most important effect was observed in the tracer lambs; all died after grazing only 18 days the two last autumns on S pasture. Selection to limit the pasture contamination permitted to prevent this outbreak in year 3. Inversely, selection so highly limited the population of Tcol during 3 years on R pasture that the absence of antigenic stimulation did not prevent half of the tracer lambs to die with 20,000 worms of Tcol nor R rams to collect 15,000 worms. This too extreme situation is possibly abnormal, but a new population can be rebuilt from a low level of larval infection of the pasture and low egg output in 1 or 2 months with favourable climatic conditions difficult to forecast. Our experiment was conducted with a limited number of species, and this limited diversity in nematode community is in accordance with theories and examples demonstrating that stability and limited high levels of infections are more related with high diversity (Silvestre et al., 2000). 5. Conclusions A selection on reducing T. circumcincta egg output permitted to limit the infection with this species with a lower efficiency compared with the selection on low T. colubriformis egg output. In this case, the species nearly disappeared, preventing outbreaks observed on
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pasture with susceptible rams, but a new population can be rebuilt in some months at a dangerous level. Selection on resistance to this species also gave a good protection to H. contortus.
Acknowledgements We thank J. Bouix from the INRA “Amélioration Génétique des Animaux” unit, for his valuable discussions necessary to elaborate the experiment. We are grateful to the technical assistance of the team of the experimental farm ‘La Sapiniere’ where the INRA 401 sheep were produced and selected. We are also grateful to J. Auger, T. Chaumeil, and P. Vincent for their technical participation for the management of the experiment at INRA Nouzilly.
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