Effects of single or trickle Haemonchus contortus experimental infection on digestibility and host responses of naïve Creole kids reared indoor

Veterinary Parasitology 191 (2013) 284–292

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Effects of single or trickle Haemonchus contortus experimental infection on digestibility and host responses of naïve Creole kids reared indoor J.C. Bambou a,∗ , W. Cei a , S. Camous b , H. Archimède a , A. Decherf a , L. Philibert a , C. Barbier c , N. Mandonnet a , E. González-García d a

INRA, UR0143 Unité de Recherches Zootechniques, Domaine Duclos, 97170 Petit-Bourg, Guadeloupe INRA, UMR1198 Biologie du Développement et Reproduction, F-78352 Jouy-en-Josas, France INRA, UE1294 Plateforme Tropicale d’Expérimentation sur l’Animal, Domaine Duclos, 97170 Petit-Bourg, Guadeloupe d INRA, UMR868, Systèmes d’Elevage Méditerranéens et Tropicaux (SELMET), Bâtiment 22, 12 Campus SupAgro-INRA, 2 Place Pierre Viala, 34060 Montpellier Cedex 1, France b c

a r t i c l e

i n f o

Article history: Received 23 December 2011 Received in revised form 5 July 2012 Accepted 18 September 2012 Keywords: Haemonchus contortus Goats Intake Immunity Digestibility

a b s t r a c t The objective of this study was to compare the effects of the type of Haemonchus contortus experimental infection (trickle infection, TI versus single infection, SI) on feed intake, nutrients digestibility, parasitological and haematological measures, and plasma leptin in Creole kids. The animals were infected over 2 periods (challenge 1 and challenge 2) of 6 weeks each, corresponding respectively to the primary and the secondary infection. Periods prior infection (1 week each) were considered as controls. The primary infection was realized with 35 Creole kids (18.40 ± 3.76 kg BW) housed in individual boxes and fed a hay-based diet. The secondary infection continued with 29 kids (21.90 ± 3.40 kg BW) from the initial 35. A total of 6 kids and 8 kids were slaughtered for measuring nematode burden at the end of the primary and the secondary infection, respectively. Measurements of nutrients digestibility were made at 0, 3 and 5 weeks post-infection for both challenges. Faecal egg count (FEC), blood eosinophilia and packed cell volume (PCV) were monitored weekly. Feed intake (dry matter intake, DMI) and nutrients digestibility were negatively affected by H. contortus infection only during the primary infection. Plasma leptin changed significantly over time (P = 0.0002) but was not affected by the infection type. Effect of infection type was observed only on crude protein digestibility during the primary infection, which was lower in the TI group (P < 0.01). The overall level of blood eosinophilia was significantly higher in the TI group (P < 0.0001) during both challenges. The overall FEC mean was significantly higher in the SI compared with the TI groups, during both challenges (P < 0.02). These results were related to the mean female length significantly higher in the SI group compared with the TI group during challenge 1 (P = 0.004), and the number of adult nematode significantly lower in the TI group compared with the SI group during the challenge 2 (P = 0.05). The results showed that the response of Creole kids to H. contortus experimental infection was in part dependent on the type of experimental infection. Our data suggest that plasma leptin would not be involved in the response of Creole kids against H. contortus infection, as no relationship between its plasma level and the transient reduction in voluntary feed intake observed in both groups during the primary infection was observed. © 2012 Elsevier B.V. All rights reserved.

1. Introduction ∗ Corresponding author at: INRA-URZ, Prise d’eau, 97170 Petit-Bourg, Guadeloupe. Tel.: +33 590 25 54 38; fax: +33 590 25 59 36. E-mail address: [email protected] (J.C. Bambou). 0304-4017/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.vetpar.2012.09.026

Gastrointestinal nematodes (GIN) are a major cause of economic loss in most small ruminant production systems throughout the world. In rural community, chemotherapy

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is no longer efficient in gastrointestinal nematodes control because of dearth of veterinary services and high cost of drugs when available. Furthermore, in developed countries due to the emergence of anthelmintic resistance (Jackson and Coop, 2000; Papadopoulos, 2008) and public concern about chemical residues in animal products, alternative control strategies are needed. It has been shown that improved nutrition is one of the most promising and feasible alternatives to chemotherapy (Coop and Kyriazakis, 1999). However, most of our understanding about the effects of gastrointestinal parasites on ruminant nutrition derived from studies in sheep. In goats, little is known about the mechanisms underlying the relationship between nutrition and gastrointestinal parasitism. Moreover, it has been demonstrated that animals acquire immunity to GIN parasites following repeated infections, so that different reaction efficiency are expected between naïve and previously infected animals, even when they were submitted to the same plane of nutrition and parasites challenge. Recently, the involvement of the adipocyte hormone leptin as a link between the immune response and the negative impact on nutrition during nematode infection in small ruminant has been suggested (Greer et al., 2005; Zaralis et al., 2008). In a previous study we demonstrated the negative effects of Haemonchus contortus infection on feed intake (dry matter intake, DMI) and digestibility of nutrients in Creole kids during an experimental infection with a single dose of infective larvae (L3) (Bambou et al., 2009a). However, the underlying mechanisms that induce reduction in voluntary feed intake in H. contortus infected kids at early and late stages of the disease have not been investigated yet. The aim of this study was to compare the effects of multiple doses (trickle infection, TI), which better mimic natural infection condition, and a single dose (single infection, SI) of H. contortus infective larvae (L3), on the digestibility and some aspects of the host response of Creole kids. We monitored DMI, nutrients digestibility, parasitological measures and plasma leptin concentration during a primary (challenge 1) and secondary (challenge 2) infection with L3 of H. contortus. 2. Materials and methods The experiment was conducted at Institut National de la Recherche Agronomique (INRA) Animal Production Unit, Guadeloupe (French West Indies) (16◦ 20 latitude North, 61◦ 30 longitude West). All animal care, handling techniques, procedures as well as license for experimental infection and blood sampling were approved by INRA, according to the certificate number A-971-18-01 of authorization to experiment on living animals issued by the French Ministry of Agriculture, before the initiation of the research. 2.1. Animals, management and experimental design The study was carried out with growing male 11months old Creole kids during two consecutive periods of 7 weeks: primary infection (challenge 1) and secondary infection (challenge 2). There was a period of four weeks

285

between finishing challenge 1 and starting challenge 2. One week prior each infection challenge were considered as control periods. All kids were born and reared indoors at INRA-Domaine de Duclos (south of Guadeloupe) into a naturally illuminated and ventilated shed. Four weeks, before challenge 1, kids were housed in individual pens (2.0 m × 1.0 m) until the end of the study. The challenge 1 started with 35 worm free (naïve) males Creole kids (18.40 ± 3.76 kg BW; 11 months old). After 7 weeks of infection kids were drenched with levamisole (Polystrongle, Coophavet, Ancenis, France, 8 mg/kg) and then were housed under worm-free conditions four weeks before the start of the challenge 2. The challenge 2 continued with 29 kids (21.90 ± 3.40 kg BW; 14 months old) from the initial 35. A total of 6 kids (n = 3 kids from the single infection, SI group and n = 3 kids from the trickle infection, TI group) were slaughtered to measure nematode burden at the end of challenge 1 (7 weeks post-infection, WPI) and 8 kids (n = 4 kids from the SI group and n = 4 kids from the TI group) at the end of challenge 2 (7 WPI). At the first day of each challenge or during the ten first days and before the morning meal (0730) we individually challenged all the kids with either a single dose of 10,000 L3 (SI) or a dose of 1000 L3/days (TI) of H. contortus. The L3 were obtained 42 days before challenge from cultures of faeces taken from anthelmintic-susceptible strain harvested from feces of monospecifically infected donor Creole goats with isolates previously obtained from Creole goats reared on pasture in different farms in Guadeloupe (Bambou et al., 2008). During the whole experiment animals received a diet composed by ad libitum 75-days-old Dichantium spp. hay and restricted concentrate (100 g d−1 ). The measurements of intake and digestibility corresponded to 0, 3 and 5 WPI after each challenge. The measurement of feed intake, BW changes, faecal eggs count (FEC) and blood parameters were performed weekly during the whole experiment. 2.2. Feed intake and in vivo total-tract digestibility Total feces collection (with faecal trays placed behind the kids) and ad libitum forage supply method was used. Pooled samples of hay, supplementary concentrate and faeces (10% per day of the wet weight) were also daily collected for chemical analyses (results shown in Table 1). For each digestibility measurement week, a 5-days period for sample collection was carried out. Offered and refused feed were individually recorded weekly in order to determine voluntary DMI and to also individually offer 115% of that respective value the week during. The in vivo apparent total-tract DM (DMD), OM (OMD), CP (CPD), NDF (NDFD) and ADF (ADFD) digestibilities were determined. 2.3. Faecal eggs count (FEC) and blood sampling To determine FEC, faecal samples of approximately 10g were weekly collected during experimental infection directly from the rectum of each kid. The faeces were kept in plastic tubes to avoid contamination and immediately transported to the laboratory in refrigerated vials. All samples were individually analysed using a modified McMaster

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Table 1 Average determined chemical composition (%, DM basis) of diet components. Haya

Concentrateb c

d

Challenge 1

DM OM CP NDF ADF ADL Hemicellulose Cellulose a b c d e f g h

Challenge 2

WPI−1e

WPI 0f

WPI 3g

WPI 5h

WPI−1

WPI 0

WPI 3

WPI 5

94.95 90.82 8.69 77.22 42.58 5.72 34.80 36.85

95.10 90.86 8.71 77.19 42.67 5.79 34.52 36.88

94.23 89.87 6.35 78.02 41.77 5.37 36.25 36.40

94.87 90.50 8.72 76.75 41.93 5.50 34.82 36.43

95.18 91.05 8.26 76.98 42.85 6.77 34.78 35.82

95.22 91.13 8.30 77.49 42.72 6.81 34.76 35.91

95.23 93.07 4.66 77.74 40.74 5.75 37.00 35.00

93.86 91.85 5.06 79.65 42.37 6.47 37.28 35.90

95.94 95.25 18.11 16.73 3.56 0.93 – –

Hay of 75-d-old Dichantium spp. grass, with forage grown with irrigation and inorganic fertilization. Offered daily in a fixed individual amount (100 g/d) as supplement. Primary infection; considered as acquisition of immunity period (challenge 1) for the naïve kids; 7 weeks in length. Secondary infection; considered as expression of immunity period (challenge 2); 7 weeks in length. Week post-infection number −1: one week before experimental challenge with H. contortus L3. Week post-infection number 0: first week after experimental challenge with H. contortus L3. Third week after experimental challenge with H. contortus L3. Fifth week after experimental challenge with H. contortus L3.

method for rapid determination and FEC was expressed as the number of eggs/g faeces. Blood samples were individually collected once a week by jugular venipuncture from each Creole kid by using disposable syringes and 20-Ga needles. A 2.5-mL portion of each blood sample was placed in commercial anticoagulant tubes (ethylenediamine tetraacetic acid K3 , EDTA tubes; Becton Dickinson, Plymouth, UK). Blood samples previously placed in EDTA coated tubes were used to measure the number of circulating eosinophils according to the method of Dawkins et al. (1989) and counted with a Malassez cell counter. The packed cell volume (PCV) was measured using the capillary microhaematocrit method. 2.4. Worm counts For both challenges, at 7 WPI kids were necropsied and the abomasum was isolated with its contents. The abomasums were opened along the greater curvature and the contents stored in 5% formalin for total worm counts in 250 ml containers. Each abomasum was then thoroughly washed with warm 0.9% NaCl to detach any adherent nematodes and the washings added to the respective animal’s abomasal contents. Parasites were then collected, counted and sorted according by sex and maturation (immature versus adult). Twenty percent of the total adult female worms from each kid were measured with a calibrated ocular scale. 2.5. Chemical analyses and analytical procedures Dry matter contents of both hay and faeces samples were determined by drying to a constant weight at 65 ◦ C in a ventilated drying oven. The samples were then ground (0.75 mm) prior to chemical analysis. The near infrared spectroscopy (NIRS) technique was used to determine the chemical composition of each sample: the following values were determined: organic matter (OM), crude protein (CP), and the proportion of cell wall components, including neutral detergent fibre (NDF) and acid detergent fibre

(ADF). Absorbance spectra of samples were recorded using a Foss NIRSystem 6500 monochromator. The databank of reference absorption spectra was established on the basis of seven tests conducted at the Animal Production Research Unit in Guadeloupe (Fanchone et al., 2007). 2.6. Leptin radioimmunoassay Plasma concentrations of leptin were quantified using the double-antibody leptin radioimmunoassay procedures described by Delavaud et al. (2000) with some modifications. Standard (0.75–60 ng recombinant ovine leptin/mL) or unknown samples (100 ␮L in routine use) were incubated with a leptin primary antiserum (Ab 7137; 50 ␮L) diluted to 1:2200 (final dilution 1:17,600) in the incubation buffer with 0.01 M EDTA containing 1% normal rabbit serum. After an initial incubation for 24 h at room temperature in a volume of 300 ␮L, 100 ␮L of 125 I-ovine leptin (diluted in the same buffer without EDTA) were added to each tube and incubation continued for 24 h at room temperature. After 48 h incubation at room temperature, the tubes were placed at 4 ◦ C until the precipitation procedure. Bound and free ligands were separated by addition of 100 ␮L of anti-rabbit IgG antiserum diluted (1:5) either in bovine serum for standard curves or in buffer for unknown samples. After mixing and resting 30 min at room temperature, 2 mL 6.25% polyethylene glycol 6000 were added and tubes were centrifuged (2740 × g; 4 ◦ C; 35 min). Assay sensitivity was 1.5 ng/mL and the intra-assay coefficient of variation ranged between 5.0 and 9.7% for different samples of control plasma. 2.7. Calculation and statistical analysis Data (PCV, eosinophilia, FEC, nutrients digestibility, body weight gain, parasite burden) were analysed by using PROC MIXED of SAS (v. 9.1, SAS Inst. Inc., Cary, NC, 2003) considering the immunity period (challenges 1 and 2), the infection type (IT), the WPI and their interactions as fixed effects when significant. The FEC and eosinophilia

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287

Table 2 Least squares means of DMI and apparent total tract digestibility of Creole kids before and after the primary challenge (challenge 1) with an oral single infection (SI) of 10,000 third-larvae stage (L3) of Haemonchus contortus or a trickle infection (TI) of 1000 L3/day during first 10 consecutive days. Item

WPI−1a e

TI h

DMI, g/d 489 Digestibility (g/kg of) i DM 692 j OM 714 k CP 473 l NDF 702 m ADF 712 n BW, kg 18.6 a b c d e f g h i j k l m n

WPI 0b

WPI 3c

WPI 5d

P-value

SI

TI

SI

TI

SI

TI

SI

SEM

WPI

ITg

IT × WPII

491

495

488

442

450

486

522

20.0

0.011

0.49

0.61

696 717 482 698 709 17.7

689 710 465 696 708 18.8

703 723 498 710 720 18.0

681 701 554 669 655 18.7

674 694 545 659 652 18.3

660 669 517 641 631 19.7

681 691 562 663 656 19.2

0.0001 0.0001 0.0001 0.0001 0.0001 0.001

0.07 0.07 0.01 0.13 0.074 0.34

0.08 0.06 0.04 0.08 0.20 0.95

f

6.4 6.1 8.1 7.9 7.5 0.8

Week post-infection (WPI) number −1: one week before experimental challenge with H. contortus L3. Week post-infection (WPI) number 0: week immediately after experimental challenge with H. contortus L3. Third week after experimental challenge with H. contortus L3. Fifth week after experimental challenge with H. contortus L3. Trickle infection with 1000 H. contortus L3/day during 10 days. Single infection with 10,000 H. contortus L3 the first day of the challenge. Infection type: single or trickle infection. Dry matter intake. Dry matter. Organic matter. Crude protein. Neutral detergent fibre. Acid detergent fibre. Body weight.

variables were log transformed in order to normalize the variances. For all traits, the experimental unit was considered the kid as they were individually fed and managed and was included in the model as a random effect. Significance was declared at ≤5% of probability; comparisons between means were tested by the least squares means procedure with adjustment for multiple comparisons (Tukey–Kramer). 3. Results 3.1. Chemical composition of the feed The chemical composition of the hay and concentrate for both experimental periods is shown in Table 1. The quality of the hay was relatively homogenous during the whole experiment, except for CP for which a greater content was observed during the challenge 1 compared with the challenge 2 but values of cell wall components (NDF, ADF, ADL) did not change. 3.2. Feed intake, digestibility and plasma leptin concentration All the parameters were monitored from one week before both experimental infections (WPI−1), which was considered as pre-infection control periods. During the primary infection (challenge 1), no significant differences were observed between WPI−1 and WPI 0. The DMI decreased significantly (P = 0.011, Table 2) in both group only during the third week post-infection (WPI). The DMI was not affected by the infection type (IT) and the interactions IT × WPI. During the secondary infection (challenge 2), no effect of WPI, IT and interaction IT × WPI was observed on DMI (Table 3). The DMI increased between

challenge 1 and 2, according to the increase in feed intake capacity of growing kids (kids were in average 4.0 kg heavier in challenge 2 compared with challenge 1, Tables 2 and 3). A significant effect of WPI was observed during both challenges on nutrients digestibility except crude protein (CP) digestibility which did not change during challenge 2 (Tables 1 and 2). Effect of IT was observed only during challenge 1, with a tendency of DM, OM and ADF average digestibility lower in kids challenged with a trickle infection (TI; P < 0.074, Table 2). A lower average digestibility of CP was observed in kids challenged with a TI during challenge 1 (P = 0.01, Table 1), but this effect was not observed during challenge 2 where no effects of IT were found (Table 2). A significant effect of IT × WPI interaction on CP digestibility was also observed only during challenge 1 (Tables 1 and 2, P = 0.04 and 0.94, respectively). No difference in plasma leptin between the preinfection periods (WPI−1, WPI 0) and the infection periods (from WPI−1 to WPI 7) was observed for both challenges (Fig. 1). Plasma leptin was analysed taking into account or not the DMI of the kids in each group. Both statistical analyses showed that plasma leptin was not affected by the infection type and changed significantly over time (P = 0.0002, Fig. 1). No significant difference was observed between challenge 1 and 2.

3.3. Blood and parasitological measures The PCV values significantly decreased during the 2 challenges in both groups until 4 WPI (P < 0.0001, Fig. 2). During challenge 1, at 2, 3 and 4 WPI the PCV value decreased more for kids in the SI group (P < 0.05, Fig. 2). Thereafter, no difference was observed between groups. The PCV increased significantly (P < 0.0001) in both groups from 4 to 7 WPI in challenge 1 and from 5 to 7 WPI in

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Table 3 Least squares means of DMI and apparent total tract digestibility of Creole kids before and after the secondary challenge (challenge 2) with an oral single infection (SI) of 10,000 third-larvae stage (L3) of Haemonchus contortus or a trickle infection (TI) of 1000 L3/day during first 10 consecutive days. WPI−1a

Item

e

TI h

DMI, g/d 554 Digestibility (g/kg of) i DM 649 j OM 665 k CP 598 l NDF 647 m ADF 617 n BW, kg 21.7 a b c d e f g h i j k l m n

WPI 0b

WPI 3c

WPI 5d

P-value

SI

TI

SI

TI

SI

TI

SI

SEM

WPI

ITg

IT × WPI

558

548

565

571

562

562

573

19.4

0.91

0.77

0.90

664 669 596 645 618 21.4

654 661 592 635 606 22.1

665 671 598 648 621 21.7

668 673 598 652 644 22.3

668 673 607 698 633 22.1

638 643 582 612 586 22.9

630 637 584 595 561 22.4

0.0031 0.0038 0.20 0.0014 0.0001 0.001

0.88 0.88 0.52 0.77 0.57 0.39

0.62 0.71 0.94 0.48 0.35 0.97

f

6.7 6.9 7.5 7.4 7.2 0.9

Week post-infection (WPI) number −1: one week before experimental challenge with H. contortus L3. Week post-infection (WPI) number 0: week immediately after experimental challenge with H. contortus L3. Third week after experimental challenge with H. contortus L3. Fifth week after experimental challenge with H. contortus L3. Trickle infection with 1000 H. contortus L3/day during 10 days. Single infection with 10,000 H. contortus L3 the first day of the challenge. Infection type: single or trickle infection. Dry matter intake. Dry matter. Organic matter. Crude protein. Neutral detergent fibre. Acid detergent fibre. Body weight.

10 9 8 7 6 5 4 3 2 1 0

Single infecon

Challenge 1

-1

0

during the challenge 1 and 2-fold higher (P = 0.023) during the challenge 2. No interaction was observed between infection type and time (WPI) during the challenge 1 but a significant interaction (P = 0.0079) was observed during the challenge 2. At the end of both challenge, the abomasal parasite burdens were estimated (n = 6 during challenge 1 and n = 8 during challenge 2, Table 4). During challenge 1, no significant difference was observed between groups in the adult nematode burden but the mean female length was significantly higher in the SI group compared with the TI group (20.3 ± 0.5 mm versus 18.0 ± 0.5 mm, P = 0.004). The immature nematode burden was significantly higher in the TI group (P = 0.02). This difference between TI and SI groups was marked in the number of immature female (P = 0.01) and a tendency was observed in the number of immature male (P = 0.06). In contrast, no significant difference in the immature nematode burden was observed during

Leptin (ng/ml)

Leptin (ng/ml)

challenge 2 (Fig. 2). A significant interaction of IT × WPI was observed in challenge 1 (P = 0.034, Fig. 2) but not in challenge 2. Blood eosinophilia significantly increased during infection and showed a peak between 2 and 5 WPI in both groups during the challenge 1 and between 2 and 4 WPI in both groups during the challenge 2 (P < 0.0001; Fig. 3). The overall level of blood eosinophilia was significantly higher in the TI group (P < 0.0001) during the 2 challenges and the significant difference according to the infection type was highlighted in challenge 2. The FEC remained at zero until 3 WPI in both groups during the challenge 1 and 4 WPI during challenge 2 (Fig. 4). A peak was observed between 4 and 5 WPI during challenge 1 in both groups. During challenge 2, a peak was observed between 5 and 6 WPI in the SI group and at 7 WPI in the TI group. When compared to the TI group, the overall FEC mean was 3-fold higher (P = 0.004) in the SI group

Trickle infecon

1

2

3

4

Weeks post-infection

5

6

10 9 8 7 6 5 4 3 2 1 0

Single infecon

Challenge 2

Trickle infecon -1

0

1

2

3

4

5

6

Weeks post-infection

Fig. 1. Means of plasma leptin (ng/ml ± SEM) according to the experimental groups: () SI, single infection (10,000 H. contortus L3 at week 0) and () TI, trickle infection (1000 H. contortus L3/day during first 10 consecutive days) after two successive challenges (challenge 1 and challenge 2).

35

35

33

33

31

31

29

29

27

27

25

PCV, %

PCV, %

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Challenge 1

23 21 19 17

Trickle infection

1 2

-2

-1

0

23

WPI: P < 0.0001 IT: P = 0.67 WPIxIT: P = 0.45

19 17

Single infection 1

2

3

Weeks post-infection

4

Trickle infection

15

15 -3

Challenge 2

25

21

WPI: P < 0.0001 IT: P = 0.088 WPIxIT: P = 0.034

5

6

289

-3

7

-2

-1

0

Single infection 1

2

3

4

Weeks post-infection

5

6

7

WPI, weeks post-infection IT, infection type (single infection or trickle infection)

Fig. 2. Means of packed cell volume (PCV) according to the experimental groups: () SI, single infection (10,000 H. contortus L3 at week 0) and () TI, trickle infection (1000 H. contortus L3/day during first 10 consecutive days) after two successive challenges (challenge 1 and challenge 2). WPI, weeks post-infection; IT, infection type (single infection or trickle infection).

Trickle infection

2500

3000

Single infection

Eosinophilia (cells x 103/ ml)

Eosinophilia (cells x 103/ ml)

3000

WPI: P < 0.0001 IT: P < 0.0001 WPIxIT: P < 0.0001

2000 1500

Challenge 1

1000 500 0 -2

-1

0

1

2

3

4

5

6

Weeks post-infection

2

2500

Single infection

WPI: P = 0.0003 IT: P < 0.0001 WPIxIT: P = 163

2000

Challenge 2

1500 1000 500 0

-3

1

Trickle infection

7

-3

-2

-1

0

1

2

3

4

5

6

7

Weeks post-infection

WPI, weeks post-infection IT, infection type (single infection or trickle infection)

Fig. 3. Means of number of blood eosinophils/ml according to the experimental groups: () SI, single infection (10,000 H. contortus L3 at week 0), () TI, trickle infection (1000 H. contortus L3/day during first 10 consecutive days) after two successive challenges (challenge 1 and challenge 2). WPI, weeks post-infection; IT, infection type (single infection or trickle infection).

the challenge 2. However, the adult nematode burden and the number of male and female nematode was significantly lower in the TI group compared with the SI group during the challenge 2 (36 ± 13 versus 302 ± 31, 7 ± 5 versus 103 ± 15 and 13 ± 6 versus 139 ± 18, P = 0.05, respectively). 4. Discussion Numerous studies aimed to investigate the physiopathology of H. contortus infection in small ruminant are based on experimental infection either with a single dose of infective L3 larvae (single infection, SI) or with multiple dose of L3 over time (trickle infection, TI) which better mimic the natural infection at pasture. However, few studies investigated the effect of the type of infection (SI versus TI) on the physiopathology of H. contortus infection

in sheep, and even less in goats (Perez et al., 2003; Shakya et al., 2011). In this study we investigated the effect of the type of infection on some aspects of the physiopathology of H. contortus infection in naïves Creole kids reared indoor. The results obtained, at first sight, seem to support our hypothesis. Indeed, primary and secondary H. contortus infections resulted in a higher FEC in single infected animals compared to the trickle infected one. A longer prepatent period and a later decrease in PCV were observed during the secondary challenge for both groups, which could suggest the development of immunity against H. contortus infection. However, no difference in the FEC between the primary and the secondary infection within groups did not support this hypothesis. This result was not consistent with our previous study showing that Creole kids are capable of developing and expressing good acquired

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Trickle infection

1000

1200

Single infection

WPI: P < 0.0001 IT: P = 0.023 WPIxIT: P = 0.09

800

FEC, eggs/g of faeces

FEC, eggs/g of faeces

1200

600 400

Challenge 1 200

WPI: P < 0.0001 IT: P = 0.004 WPIxIT: P = 0.0079

800 600 400

Challenge 2

0 -3

2

1000

Single infection

200

0

1

Trickle infection

-2

-1

0

1

2

3

Weeks post-infection

4

5

6

7

-3

-2

-1

0

1

2

3

Weeks post-infection

4

5

6

7

WPI, weeks post-infection IT, infection type (single infection or trickle infection)

Fig. 4. Geometric means of faecal egg counts (FEC) according to the experimental groups: () SI, single infection (10,000 H. contortus L3 at week 0), () TI, trickle infection (1000 H. contortus L3/day during first 10 consecutive days) after two successive challenges (challenge 1 and challenge 2). WPI, weeks post-infection; IT, infection type (single infection or trickle infection). Table 4 Least squares means of adult and immature nematode counts at week seven after primary infection (challenge 1) and secondary infection (challenge 2) of Creole kids with an oral single infection (SI) of 10,000 third-larvae stage (L3) of Haemonchus contortus or a trickle infection (TI) of 1000 L3/day during first 10 consecutive days. Challenge 1a TIc Female Male Nematode burdene Immature female Immature male Immature burdenf Female length (mm) a b c d e f

361 377 805 131 48 182 18.0

Challenge 2b SId

± ± ± ± ± ± ±

39 39 69 18 10 24 0.5

256 212 512 6 2 9 20.3

± ± ± ± ± ± ±

33 29 55 4 2 5 0.5

P value

TI

0.55 0.34 0.45 0.01 0.06 0.02 0.004

13 7 36 4 1 5 18.6

SI ± ± ± ± ± ± ±

6 5 13 3 1 4 0.4

139 103 302 27 11 39 17.6

P value ± ± ± ± ± ± ±

18 15 31 6 4 8 0.4

0.05 0.05 0.05 0.22 0.22 0.21 0.07

Challenge 1, primary infection; considered as acquisition of immunity period for the naïve kids; 7 weeks in length. Challenge 2, secondary infection; considered as expression of immunity period; 7 weeks in length. Trickle infection with 1000 H. contortus L3/day during 10 days. Single infection with 10,000 H. contortus L3 the first day of the challenge. Total adult nematode. Total immature nematode.

immunity measured by a decrease in FEC, during a secondary infection with H. contortus preceded by a single primary infection (Bambou et al., 2009a). Previous experiments with non-naïves Creoles and West African Dwarf goat kids, observed a more pronounced anaemia associated with a higher FEC, than that observed in the present study (Bambou et al., 2009b; Fakae et al., 1999). However, herein the host response to infection, as measured by heamatocrit and eosinophilia significantly changed with time. This suggests that the experimental infections conducted in the present study inducing a sub-clinical rather than a marked H. contortus infection, failed to induce a high degree of protection against further infections. Thus, when we compared our results in worm counts during the primary infection with previous studies in kids and lambs, the worm establishment rates were numerically lower (Bricarello et al., 2004; Chiejina et al., 2010; Fakae et al., 1999). Statistical differences between SI and TI groups in worm burden were observed only in the immature worm burden and adults female length during the primary infection. The higher number of immature worm in the TI group

could suggest a regulation of the worm population, but this hypothesis needs further investigations. The lower adult female length observed in the TI group compared with the SI group is consistent with the response to infection measured by FEC. Indeed, it has been shown in sheep that worm female length is significantly positively correlated with FEC and thus with fecundity (Stear and Bishop, 1999; Terefe et al., 2005). In contrast, the decrease in the adult nematode counts observed in both groups during the secondary infection was not related with a decrease in FEC or an increase in worm female length. This result could be due to the low number of kids slaughtered at each point, but it could also be related to the complexity of the relationship between the biology of the nematode population, the induced pathogenic process and the host response. The host response as measured by the circulating eosinophilia, was significantly higher in the TI group during both challenges. Whereas in the SI group a peak was observed between 2 and 3 WPI, in the TI group the circulating eosinophilia was increased during both challenges. Furthermore, circulating eosinophilia observed in

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both groups was higher during the secondary infection. These data are in keeping with previous studies showing that recruitment of eosinophils is more rapid and more pronounced locally in the abomasal mucosa and at the peripheral level in kids and lambs previously infected (Balic et al., 2002; Bambou et al., 2009a; Fakae et al., 1999; Lacroux et al., 2006). These results are consistent with the role of eosinophils, as immune effector cells specifically directed against larval rather than adults stages of gastrointestinal nematode (Balic et al., 2006; Robinson et al., 2010; Terefe et al., 2007). A significant transient reduction in voluntary feed intake, as measured by a depression of DMI, was observed only during the third week of the primary infection. The lack of such an effect during the secondary infection was consistent with our previous findings in the same experimental model (Bambou et al., 2009a). In contrast with Teladorsagia circumcincta and Trichostrongylus colubriformis infections, which are known for a long time to induce a persistent reduction in voluntary feed intake throughout the course of parasitic challenges, the effect of H. contortus on feed intake is much less pronounced (Coop et al., 1977, 1982; Kyriazakis et al., 1996). Thus, in accordance with our previous study, the effect of H. contortus infection on growth rate observed here could be related to the decrease in nutrients digestibility (Bambou et al., 2009a). As previously discussed (Bambou et al., 2009b), in the context of pathogenic challenges, the CP is the most critical nutrient because of the proteinaceous nature of many components of the immune system, such as immunoglobulins, mucoproteins, and cellular mediators, such as chimiokines. During both infections, a slight statistical decrease of OM digestibility was observed in both groups. However in contrast with our previous findings, CP digestibility increased during the primary infection and did not change during the secondary infection (Bambou et al., 2009b). This result probably related with the sub-clinical H. contortus infection induced in the present study, suggested that the impact of gastrointestinal parasitism on nitrogen metabolism would be correlated with the level of infection. This hypothesis needs further investigation to be validated. Recently, the role of plasma leptin in the control of voluntary feed intake in nematode infected sheep and lambs has been investigated. Initially, it has been suggested that the reduction of voluntary feed intake would be related to the development of immunity against nematode infection rather than the damage caused by the parasite on the intestinal mucosa (Greer et al., 2005). Then, it has been showed that plasma leptin increased significantly during experimental infection with T. circumcincta and T. colubriformis (Greer et al., 2009; Zaralis et al., 2008, 2009). In contrast, in the present study the level of plasma leptin did not change over time in both groups. In our study we did not compare our results from infected kids with a non-infected control group, as it has been done in the cited references. However, plasma leptin was measured from one week before and at the day of the experimental infection. The levels of plasma leptin before and during infections were not different. Our data suggest that in H. contortus infected kids the transient reduction of voluntary feed intake and the decrease in the ADG were not related

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with plasma leptin. Even if an increase in plasma leptin has been observed in sheep, all the studies concluded that reduction in voluntary feed intake observed in T. circumcincta and T. colubriformis infected sheep, was not related to plasma leptin. In conclusion, the data showed that the response of Creole kids to H. contortus experimental infection was dependent on the type of experimental infection (i.e. single versus trickle) and the immunological stage (primary versus secondary infections). The TI group showed lower FEC than the SI group during both infections. In the TI group, the immature worm burden was higher during the primary infection and the adult worm burden was higher during the secondary infection. A transient reduction of voluntary feed intake was observed in both groups during the primary infection but was not related to plasma leptin. The results suggest that, in the conditions of this study, plasma leptin would not be involved in the response of Creole kids against H. contortus infection.

Acknowledgements The authors want to give thanks to H. Varo and L. Philibert for their excellent technical assistance and P. Despois and F. Labirin for care and handling of animals. This work was supported in part by INRA and by La Région Guadeloupe. JCB was supported by a post-doctoral fellowship from Le Conseil Régional de la Guadeloupe. WC was supported by a doctoral fellowship from Le Conseil Régional de la Guadeloupe.

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