Direct anthelmintic and immunostimulatory effects of oral dosing semi-purified phytohaemagglutinin lectin in sheep infected with Teladorsagia circumcincta and Trichostrongylus colubriformis

Veterinary Parasitology 187 (2012) 267–274

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Direct anthelmintic and immunostimulatory effects of oral dosing semi-purified phytohaemagglutinin lectin in sheep infected with Teladorsagia circumcincta and Trichostrongylus colubriformis L. Ríos-de Álvarez a,∗ , F. Jackson b , A.W. Greer c , G. Grant d , E. Jackson b , A.A. Morrison b , J.F. Huntley b a b c d

Instituto de Producción Animal, Facultad de Agronomía, Universidad Central de Venezuela, Maracay, Edo. Aragua, Venezuela Parasitology Division, Moredun Research Institute, Edinburgh EH26 0PZ, Scotland, UK Faculty of Agriculture and Life Sciences, PO Box 84, Lincoln University, Canterbury, New Zealand Gut Immunology, RINH, Institute of Medical Sciences Buildings, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, Scotland, UK

a r t i c l e

i n f o

Article history: Received 31 October 2011 Received in revised form 19 December 2011 Accepted 2 January 2012 Keywords: PHA lectin Direct and indirect anthelmintic effect Immune-stimulation Nematoda

a b s t r a c t Lectins are plant secondary compounds that can have anthelmintic properties in vitro. In particular, the phytohaemagglutinin lectin extracted from Phaseolus vulgaris has been shown to inhibit the feeding of Trichostrongylus colubriformis and Teladorsagia circumcincta L1 larvae. However, little is known about the potential anthelmintic properties of this lectin in vivo and its suitability to control gastrointestinal parasite infections in lambs. In a 2 × 2 study, lambs were either orally dosed, or not, with 2.3 mg semi-purified PHA lectin per kg live weight (LW) per day, whilst concurrently infected, or not, with 1000 T. circumcincta and 1000 T. colubriformis L3 infective larvae per day for 42 days. There were no adverse clinical effects observed with this dose of PHA lectin. Although worm burdens were similar, animals dosed with PHA lectin had reduced concentration of nematode eggs in the faeces compared with their non-lectin dosed counterparts (P = 0.026), suggesting that there may be a direct effect of PHA lectin on parasite fecundity. Irrespective of infection, PHA lectin had immune-stimulatory properties with increased eosinophillia in both abomasal and small intestine tissue sections taken at slaughter on day 42 (P < 0.02 for both) and a tendency for decreased ability of Teladorsagia larvae to penetrate abomasal tissue explants (P = 0.06). Compared with infection alone, concurrent PHA lectin dosing and infection further increased the number of eosinophils (P < 0.01), PAS-positive (mucin-producing cells) (P = 0.03) and tended to increase the number of T helper cells (P = 0.06). No interactions were observed for cell populations in small intestine tissue sections. These results suggest PHA lectin could have two possible modes of action against T. circumcincta and T. colubriformis, a direct anthelmintic effect on nematode fecundity and an indirect effect through enhancing local immune responses in the host. © 2012 Elsevier B.V. All rights reserved.

1. Introduction

∗ Corresponding author. Tel.: +58 412 4767809; fax: +58 243 5507009. E-mail addresses: [email protected], [email protected] (L. Ríos-de Álvarez). 0304-4017/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2012.01.005

Gastrointestinal parasites are ubiquitous in grazing livestock throughout the world and represent a major impediment to efficient production systems. The desire for cost-effective and natural approaches to control nematode parasites has led to investigations into alternatives to chemical anthelmintics. In particular, plants with a

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range of bioactive properties have demonstrated potential anthelmintic effects in both in vitro and in vivo studies. In addition, plant secondary compounds, such as lectins, have been shown to resist degradation in the gastrointestinal tract and have also been reported to have a dose-dependent effect on the pathology and physiology of the intestine (Pusztai, 1991). Further, plant lectins have also been observed to have immune-modulatory effects, including stimulation of mucosal mast cells (MMC) and Tlymphocytes, increased CD4+ and CD8+ cells in both birds (Johnsen and Zuk, 1999; Kennedy and Nager, 2006; Owen ´ and Clayton, 2007; Tella et al., 2008) and mice (Karmanska ´ and Michalska, 1978; Karmanska et al., 1996) infected with parasites. Preliminary in vitro studies in our laboratory have also shown some plant lectins have a strong ability to inhibit the feeding of developing larvae (Ríos-Álvarez, 2009) and as such may confer some direct anthelmintic properties. More specifically, the kidney bean lectin phytohaemagluttin (PHA) demonstrated the lowest IC50 concentration of the plant lectins screened during these investigations, an effect which was consistent across Teladorsagia circumcincta, Haemonchus contortus and Trichostrongylus colubriformis. In addition, kidney beans have the added advantage of being widely available, and generally contain high (∼20 g per kg DM) amounts of lectin that is relatively easy to extract, is proteolytically stable and has a low toxicity (Banwell et al., 1984; Bardocz et al., 1995, 1996; Carbonaro et al., 2000; Fitches et al., 2001; Kennedy and Nager, 2006). In this study we investigated the potential direct anthelmintic effect of PHA lectin in lambs with a mixed infection of T. circumcincta and T. colubriformis.

2. Materials and methods 2.1. Experimental design Twenty-four parasite naive 4-month-old mixed sex Scottish Blackface × Texel lambs were allocated to one of four groups (n = 6) that were balanced for both sex and live weight (LW; 34.3 ± 4.2 kg). Each group was randomly allocated to one of four treatment regimes in a 2 × 2 factorial design. The first treatment factor was parasite infection, with animals either infected (+P), or not (−P), with the equivalent of 1000 infective L3 T. colubriformis and 1000 infective L3 T. circumcincta larvae per day in a three-times weekly trickle-dosing regime. Larvae were suspended in water and were administered orally on Monday, Wednesday and Friday of each week for six weeks. The second factor was lectin administration, with animals receiving either a daily dose of 200 mg of a semi-purified PHA extract (+L) or a sham treatment with tap water (−L). The extract was 40.24% PHA (w/w) and provided initial daily doses of 2.3 mg PHA per kg LW per day. Semi purified PHA lectin for all + L animals was reconstituted each morning when dosing occurred, using tap water and were administered orally via syringe five times weekly from Monday to Friday. All animals were closely monitored for any side effects following lectin administration. Treatments continued from day 0 until slaughter on day 42 of infection.

2.2. Preparation of the semi-purified phytohaemagglutinin lectin (PHA lectin) White kidney beans (Phaseolus vulgaris) were ground using a hammermill fitted with a 1 mm mesh. The seed meal (100 g) was mixed with 1 l of 0.2 M sodium acetate pH 5.0 buffer and left stirring at +4 ◦ C overnight. Large particulate matter was allowed to settle out. The upper liquid was decanted off, centrifuged at 48,000g for 20–30 min and the supernatant retained. This was mixed with ammonium sulphate to 80% of saturation and the proteins allowed to precipitate at +4 ◦ C overnight. A proportion of the clear upper liquid was decanted off and discarded. The remaining liquid/precipitated proteins were centrifuged at 48,000g for 15 min. The resultant pellet was re-suspended in water (200–300 ml), the pH of the solution adjusted to 7.0 and then dialysed extensively against distilled water. After dialysis, the pH of the solution was checked, the solution centrifuged (if necessary) and the supernatant freeze dried. The yield from 1 kg meal was approximately 45 g dry weight of albumins (contained 18.1 g PHA). 2.3. Animal feeding and sampling Animals were group housed within their treatments with access to fresh water and ad libitum access to fresh hay with a digestibility of organic matter (DOM) of 593 g DOM per kg dry matter (DM), supplying 8.5 megajoules of metabolisable energy (MJME) per kg DM and containing 89 g crude protein (CP) per kg DM in addition to 0.5 kg per day per animal of a ruminant concentrate with 692 g DOM per kg DM, supplying 11.1 MJME per kg DM and containing 179 g CP per kg DM. Live weight was recorded weekly with 24 h fasted LW recorded on day 0 and on day 42 prior to slaughter. Blood samples were collected weekly by jugular venipuncture into heparinised tubes (vacutainer systems, Becton Dickinson, UK), were centrifuged at 1200 × g for 15 min at which point the plasma was removed and stored at −20 ◦ C until analysis for plasma total protein, albumin and inorganic phosphate performed using a microcentrifugal analyser (Ilab 300 Plus, Instrumentation laboratory, Warrington, Cheshire, UK) using Instrumentation Laboratory kits cat no 18481300, 18481800 and 18481900 for total protein, albumin and phosphorus, respectively. Faecal samples were collected directly from the rectum twice weekly (Monday and Thursday) for the determination of the concentration of nematode eggs in the faeces (FEC) using the modified floatation technique (Christie and Jackson, 1982) and were expressed as eggs per g of fresh faeces (epg). From week 4, a sub-sample of the faeces collected from each infected animal was taken for coproculture, according to the methodology described by Kaufmann (1996). Briefly, 3–20 g faeces from each animal were incubated separately for 10 days at 22–23 ◦ C. Following incubation, faeces were left to soak in water for 24 h to allow the larvae to exit the faecal mass before cleaning by baermannisation. Proportional recovery of larvae was calculated by dividing the number of L3’s recovered by the number of eggs in the original sample (epg × faeces weight). From each coproculture a sub-sample of L3 was taken, exsheathed and speciated (MAFF, 1986) for the

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estimation of the proportion of T. colubriformis and T. circumcincta. All animals were slaughtered on day 42 by stunning with a captive bolt followed by exsanguination through severance of the carotid artery and jugular vein. Immediately following slaughter the abomasum and first five metres of the proximal small intestine from all animals were excised. Worm recovery from the abomasum and small intestine of infected animals was performed following incubation in physiological saline at 37 ◦ C for 4 h as described by Jackson et al. (1984). Worm enumeration according to stage of development and sex was performed from a 1% aliquot. 2.4. In vitro direct challenge (IVDC) The ability of abomasal tissue to prevent larval establishment was examined using the IVDC technique as described by Jackson et al. (2004). Briefly, fundic folds were excised from the abomasa immediately following slaughter and rinsed gently in warm physiological saline (0.85%) to remove the majority of adherent digesta. Three tissue samples measuring approximately 2 cm × 2 cm were removed from each animal and placed into a separate well of a Corning six-well plate. Warm Hank’s medium (Sigma Chemical Company, St. Louis, MI, USA) with 20 mmol per l Hepes (Boehringer Mannheim GmbH, Germany) and 2 ml phenol red was added to 1 l of sterile H2 O and brought to pH of 7.6 with NaOH, was added to surround but not submerge the tissues. The barrels of 10 ml syringes with the needle end removed were placed onto the centre of each piece of dissected tissue to provide an isolation cylinder to contain the larvae. Doses of approximately 2000 exsheathed T. circumcincta larvae were introduced in 0.5 ml of saline onto the mucosal surface of each tissue section within the isolation cylinder with sufficient pressure applied to the lid of the six-well plate to ensure an effective seal between the cylinder and tissue. The plates were then incubated at 38 ◦ C in a high oxygen concentration for 3 h. The time taken from slaughter to incubation was no more than 20 min. Following incubation the larvae loosely adhering to the tissue were retrieved by repeated plunging the explants 30 times in warm physiological saline (wash). Larvae that were closely associated with the tissue were retrieved by digesting the tissue sections by incubation in pepsin and HCl for 12 h (digest). The number of larvae in each of the wash and digest fractions were enumerated with the proportion of larvae recovered from the digest fraction indicating those which had the closest association with the mucosal surface. 2.5. Histochemical and immunohistochemical analysis Samples of abomasal and intestinal tissues from each animal were removed immediately following slaughter and fixed in either phosphate buffered saline (PBS) containing 4% paraformaldehyde (PF) for 6 h at room temperature (Newlands et al., 1984) or in Zn salts fixative at room temperature for 24 h (Gonzáles et al., 2001). After fixation, tissues were processed and embedded in paraffin; 5 ␮m sections were cut, mounted on slides and dried for 12 h at 40 ◦ C. For PF fixed sections, general

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histochemical characteristics were assessed following haematoxylin–eosin staining. Mucosal mast cell (MMC) and eosinophils were enumerated following staining with toluidine blue (Enerback, 1966) and carbol-chromotrope (Lendrum, 1944), respectively. In abomasal sections mucin-producing cells and in the intestine, goblet cells in the epithelium and Paneth cells in the crypts were detected using Periodic Acid Schiffs (PAS) (Mantle and Allen, 1978). Neutral and acidic mucins were identified through alcian blue-PAS staining (Newlands et al., 1990). Unfortunately, the Ab-PAS staining was not able to distinguish between the different types of mucin-producing cells, and almost all goblet cells appeared to be stained homogeneously with PAS indicating the presence of neutral mucin. Stained cells were enumerated using an 10× eye piece containing a calibrated graticule and an 40× objective lens. Zn salts fixed tissue sections were employed for immunostaining. Pan T cells were detected with anti-CD3 (AntiCD3, Dako, Ltd., Ely, UK) at a dilution of 1/200. T helper cells were detected using antibody 17D Ovine CD4 (isotype IgG1, Batch 160995) at a dilution of 1/1000 (Hein et al., 1987). Controls consisted of tissue sections where the primary antibody was replaced with mouse serum at a rate of 1:200 (IgG1 myeloma, clone MOPC21, Sigma catalog no. 026K4804). Stained cells were enumerated using the software ImageJ (1.41.g Wayne Rasband National Institutes of Health, USA). All cell counts from both PF and Zn salts fixed tissue sections were made systematically in the epithelium and mucosa from the mean of 10 graticule fields, and were expressed as cells per mm2 of intestinal tissue. 2.6. Statistical analysis Analyses were performed using GenStat statistical software version 7.2 (Lawes Agricultural Trust, 2004) as a 2 × 2 factorial design with lectin and infection as the factors unless otherwise stated. Prior to statistical analysis, FEC and worm burdens were log-transformed (log 10 (n + 1)). All transformed data are presented as back-transformed means unless otherwise stated. FEC, speciation and proportional recovery from coproculture and serum data underwent sequential comparison for antedependence structures prior to being analysed for repeated measures using a restricted maximum likelihood (REML) with time included as a factor. Worm burdens, overall live weight gain and intestinal tissue cell concentrations were analysed using a general ANOVA. For FEC, worm burden and coprocultures analysis was performed on data from infected animals; consequently infection was removed as a factor. In addition, FEC was analysed only from the time eggs appeared in the faeces on day 18. 3. Results 3.1. Clinical findings No adverse reactions or other clinical problems were observed at any stage in any of the animals dosed with PHA. Although some animals presented isolated cases of diarrhoea, treatments did not affect the dry matter content of the faeces with means ± s.e. of 426 ± 30.3, 414 ± 40.0,

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Backtransformed faecal egg counts (eggs/g)

6000

treatment (F1,40 = 6.26, P = 0.01) but not time (F3,40 = 2.12, P = 0.112). Only T. colubriformis and T. circumcincta larvae were recovered from the coprocultures. The mean proportion larvae identified as T. circumcincta decreased with time (F3,7 = 15.4, P = 0.001) and was not influenced by lectin treatment (F1,31 = 0.39, P = 0.54), with values being 0.68 ± 0.037, 0.38 ± 0.065, 0.30 ± 0.042 and 0.23 ± 0.041 for days 25, 31, 35 and 42 of infection, respectively.

5000 (+P-L)

4000

(+P+L)

3000 2000

3.5. Worm burdens

1000 0 0

10

20

30

40

50

Day of Infection Fig. 1. Mean backtransformed faecal egg counts (FEC; eggs per g) for parasitised animals that either were dosed with lectin (+P+L) or were not dosed (+P−L). Error bars represent 95% confidence intervals.

327 ± 24.3 and 403 ± 26.7 g per kg for −P−L, −P+L, +P−L and +P+L, respectively (F3,32 = 2.10, P = 0.12). 3.2. Live weight and plasma analysis Mean live weight gains from day 0 until day 42 were 89 ± 20.3, 93 ± 12.5, 81 ± 14.6 and 62 ± 16.4 g per day, for −P−L, −P+L, +P−L and +P+L, respectively, and were not influenced by infection or lectin treatment (F1,20 < 1.77, P > 0.05 for both). Plasma total protein, albumin and globulin mean concentrations at day 0 were 63.7 ± 1.2, 38.6 ± 0.7 and 25.1 ± 0.9 g per l, respectively. For all groups there was no effect of infection (F1,35 < 1.53, P > 0.05 for all) or lectin (F1,35 < 0.19, P > 0.05 for all) but there was an effect of time (F5,99 > 4.89, P < 0.001) that reflected a temporary decrease of each blood parameter (−6 to 9%) on day 7 only. Mean plasma phosphorous concentration for all animals at day 0 was 11.0 ± 0.5 g per l and was not influenced by time (F5,14 = 1.20, P = 0.36), infection (F1,8 = 2.02, P = 0.19) or lectin treatment (F1,8 = 0.09, P = 0.76). 3.3. Faecal egg counts Mean log 10 back-transformed FEC are shown in Fig. 1. The FEC of all infected animal increased throughout the duration of the study with the exception of a temporary decrease in +P−L animals on day 39 only. Overall, for faecal samples taken after day 18, there was an effect of time (F7,80 = 7.57, P < 0.001) and there tended to be an effect of lectin treatment (F1,80 = 3.66, P = 0.06). Re-analysis of the data excluding day 39 suggests there was an overall effect of time (F7,75 = 7.47, P < 0.001) and an effect of lectin treatment (F1,75 = 5.15, P = 0.026) but no time × lectin interaction (F6,75 = 1.08, P = 0.38). 3.4. Coprocultures and species prevalence Mean proportion of nematode eggs that were recovered as L3 larvae was 0.10 ± 0.018 and 0.27 ± 0.073 for +P−L and +P+L, respectively, which was influenced by

Mean total burdens (±s.e.) were 4416 ± 878 and 3475 ± 792 (T. circumcincta) and 6708 ± 414 and 6500 ± 295.5 (T. colubriformis) for control (+P−L) and lectin treated (+P+L) groups, respectively. Overall, 45% and 49% were adult female and 17% and 5% were juvenile for T. circumcincta and T. colubriformis, respectively. There were no significant differences in the total T. circumcincta and T. colubriformis burdens nor in the sex ratio or immature worm vs. mature worm numbers as a consequence of lectin treatment (F1,10 < 1.81, P > 0.05 for all). 3.6. In vitro direct challenge (IVDC) The proportions of larvae found to be closely associated with the tissue was reduced by infection (F1,16 = 61.0, P < 0.001) and tended to be reduced by lectin administration (F1,16 = 4.01, P = 0.06), with mean values being 68.0 ± 4.12, 57.8 ± 3.25, 19.3 ± 8.79 and 5.3 ± 2.41 per 100 larvae for −P−L, −P+L, +P−L, +P+L, respectively. 3.7. Histochemical and immunohistochemical analysis Histochemical and immunohistochemical cell counts for abomasal and small intestine tissue sections are given in Table 1. Eosinophils concentrations in abomasal tissue sections displayed a lectin × infection interaction (F1,20 = 9.16, P = 0.01) which reflected an increase in parasitised animals that was exacerbated by lectin administration. For small intestinal tissue sections, eosinophils concentrations were increased by both lectin administration (F1,20 = 7.90, P = 0. 016) and by infection (F1,20 = 4.61, P = 0.05). For MMC concentrations, there was no effect of either lectin administration or parasite infection on the numbers of MMC observed in either abomasal or intestinal tissue sections (F1,20 < 2.68, P > 0.05 for all). Mucin producing cells stained with PAS in abomasal tissue sections displayed an interaction between infection and lectin administration (F1,20 = 5.16, P = 0.03) reflecting greater numbers of PAS positive cells only in animals concurrently receiving infection and lectin (+P+L). In addition, there tended to be an interaction between parasite infection and lectin administration in Ab-PAS cells (F1,20 = 3.84, P = 0.06) which reflected infection only resulting in increase in cell concentrations in those animals that concurrently received lectin (+P+L). No significant effects of infection, lectin administration or interactions were observed for either PAS or Ab-PAS cells in the small intestine (F1,20 < 0.59, P > 0.05 for all). Pan T cell concentrations were increased due to parasite infection in abomasal tissue sections (F1,20 = 7.45,

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Table 1 Eosinophil, mucosal mast cell, PAS, Ab-PAS, Pan T and T helper cell counts (cells per mm2 ± s.e.m.) for abomasal and small intestinal tissue retrieved from animals at day 42 that were either uninfected (−P) or infected with 1000 T. colubriformis and 1000 T. circumcincta per day for six weeks (+P) whilst also receiving either 0 (−L) or 80 mg PHA lectin per day (+L). Treatment −P−L Abomasal tissue (cells per mm2 ) 0.2 ± 0.1 Eosinophils 189 ± 8.7 Mucosal mast cells PAS 80.3 ± 5.2 Ab-PAS 106 ± 5.9 46.2 ± 3.7 Pan T 9.9 ± 0.7 T helper Small intestine tissue (cells per mm2 ) 13.2 ± 4.1 Eosinophils 217 ± 9.7 Mucosal mast cells 27.8 ± 1.8 PAS Ab-PAS 37. 3 ± 1.9 Pan T 211 ± 8.8 94.3 ± 10.4 T helper

Effects −P+L

+P−L

+P+L

Lectin

Infection

Lectin × Infection

0.1 221 68.0 82.4 49.7 9.7

± ± ± ± ± ±

0.09 9.5 5.3 5.6 6.0 0.7

9.9 225 65.5 98.4 70.1 29.3

± ± ± ± ± ±

3.8 10.1 5.4 6.6 4.2 10.4

32.8 166 98.7 124 76.0 18.3

± ± ± ± ± ±

4.2 10.3 6.4 7.8 4.3 1.8

0.01 0.59 0.31 0.93 0.61 0.05

<0.001 0.68 0.44 0.19 0.01 <0.001

0.01 0.07 0.03 0.06 0.90 0.06

28.6 253 31.0 36.2 173 85.4

± ± ± ± ± ±

3.4 9.1 1.9 2.0 11.1 23.5

23.1 224 30.0 28.1 173 81.0

± ± ± ± ± ±

4.4 8.7 1.9 1.6 9.4 10.2

51.9 187 27.2 44.5 195 77.6

± ± ± ± ± ±

3.8 8.2 1.5 2.9 10 10.8

0.016 0.98 0.96 0.14 0.67 0.54

0.05 0.21 0.83 0.93 0.66 0.30

0.48 0.12 0.45 0.09 0.11 0.78

P = 0.01) but not small intestine tissue sections (F1,20 = 0.20, P = 0.66) and were not influenced by lectin administration (F1,20 < 0.26, P > 0.05 for both). For T helper cell populations, concentrations in abomasal tissue sections were

influenced by both parasite infection (F1,20 = 26.6, P < 0.001) and lectin administration (F1,20 = 4.30, P = 0.05). In addition, there was a tendency for an infection × lectin administration interaction which reflected a lesser increase due

Fig. 2. (A–F) Photographs (40×) of tissue sections showing eosinophils (A and B), PAS-positive cells (mucin-producing cells) (C and D) and T helper cells (E and F), in sections retrieved from abomasa of parasitised animals with no lectin (+P−L) (A, C, and E), compared to parasitised animals that received lectin (+P+L) (B, D, and F). Significantly greater numbers of cells were observed due to the interaction lectin × infection.

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to parasite infection in animals concurrently receiving parasites and lectin (+P+L) compared to animals receiving parasite infection alone (+P−L) (F1,20 = 3.99, P = 0.06). There were no significant effects of treatment observed for either Pan T cells or T helper cells in small intestine tissue section (F1,20 < 2.75, P > 0.05 for all). Fig. 2(A)–(F) shows photographs of tissue sections demonstrating the significant changes due to the interaction lectin × parasite infection in the population of eosinophils, PAS-positive cells (mucin-producing cells) and T helper cells from the abomasum. 4. Discussion This study was primarily designed to investigate any direct anthelmintic effects of PHA lectin on a multi species worm population, using faecal egg counts and worm burden as the key parasitological comparators. The study covered a relatively short period of first exposure to infection (6 weeks) in order to minimize the confounding effects that the acquisition of immunity can have upon egg count and worm burden. Previous studies have shown that the various mechanisms that regulate parasite fecundity, larval establishment and adult persistence (Barger, 1987) would not be expected to be operating effectively during this time frame with either of the nematode species used in the current study (Seaton et al., 1989a,b; Bown et al., 1990). However, it is evident that at slaughter on day 42 infected animals, regardless of lectin treatment, did possess an enhanced immunological response, as indicated by eosinophillia in both abomasal and small intestine tissue sections and increased concentrations of T-cells and T-helper cells in abomasal tissue sections and a reduced ability of T. circumcincta L3 larvae to penetrate the abomasal tissue sections in the in vitro direct challenge. Whether or not this level of immune development was sufficient to provide any control of the worm population, and thus confound the ability of the study to measure the direct anti-parasitic effect of PHA lectins, remains to be determined. One of the most striking results observed in the current study was the reduced concentration of nematode eggs in the faeces in animals that were administered PHA lectin, an effect that was consistent on all but one sampling occasion from day 21 of infection. Given that this difference in FEC would not be expected to be accounted for by differences in faecal output and that lectin administration did not affect either the total burden or the sex ratio of adult worms of either species, it seems reasonable to conclude this difference in FEC was due to a lower per capita fecundity in the nematodes residing in PHA lectin treated animals. Further support for this can found in a calculation of total egg production of the animals at slaughter. Taking into account the food consumption required to meet the energetic demands for maintenance and growth in the week prior to slaughter (AFRC, 1993) and assuming the proportion of T. circumcincta and T. colubriformis larvae recovered in the coprocultures reflected the proportion of eggs produced by each species, it can be calculated the administration of PHA lectin reduced the mean daily egg production per female by 12% and 32% for T. circumcincta

and T. colubriformis, respectively. On the one hand, this may reflect an enhanced immune response in lectin treated ani´ ´ mals (Karmanska and Michalska, 1978; Karmanska et al., 1996; Piekarska et al., 2008) which reduces female parasite fecundity. Alternatively, given the expected immaturity of the host immune response during this time, especially as early as four weeks post-infection, the observed and calculated differences in FEC and female fecundity may, in part, be mediated through a direct anti-worm effect. The mechanisms underpinning this direct anthelmintic activity are not clear but might possibly be due to changes in behaviour induced by lectin binding that affected feeding, chemosensory or egg laying activity of the nematodes. Further, it may be anticipated these effects of lectin may be concentration dependent. In rats, PHA has been reported to strongly bind to the brush border in the small intestine and stomach (Pusztai et al., 1990; Bardocz et al., 1995; Linderoth et al., 2006). Whilst it seems reasonable to assume that the concentrations of PHA in the abomasum and intestine were sufficiently high to produce the apparent reductions in parasite fecundity, the actual concentrations achieved in the gastrointestinal tract, and the proportion of lectin, which remains freely available to interact with the larvae, remains unknown. There was no evidence of any effective immunoregulation of the infrapopulation in this study, which is not surprising given the known complexity of these mechanisms (Jackson and Miller, 2006). However, allowing for the acknowledged immunostimulatory properties of lectins ´ ´ (Karmanska and Michalska, 1978; Karmanska et al., 1996; Lavelle et al., 2000; Piekarska et al., 2008) one cannot discount the possibility that there may have been some immunological changes induced by lectin treatment that might have subsequently influenced the rate of acquisition and expression of immunity. Evidence for such effects in the current study can be observed from the greater level of eosinophillia in both the abomasum and small intestine, greater concentration of PAS cells in the abomasum and a tendency for more Ab-PAS cells in tissues of animals concurrently infected and given PHA lectin compared with their counterparts that were infected alone (Table 1). These observations are comparable with those reported in rats whereby treatment with PHA lectin increased the number of mucin-containing cells in the intestine during repair after damage or trauma (Grant et al., 2008). Nevertheless, it is difficult to directly extrapolate these findings in rats to the present studies in sheep, with differences in species, parasite and dose of PHA. On the other hand, there is also a tendency in the current study for animals concurrently infected and administered PHA lectin to have reduced Thelper and mast cell concentrations in abomasal tissue. Although the impact of such changes in cell populations on the parasite population cannot be fully determined, the fact that PHA lectin treatment did not affect the worm burden suggests that this effector arm of the immune response was not influenced. However, with this in mind, nearly 95% of L3 T. circumcincta larvae failed to penetrate the abomasal tissue of PHA lectin treated and infected animals during the IVDC, compared with 81% in infected animals not administered lectin. This difference in the ability of larvae to associate with the mucosal tissues is possibly a

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reflection of the enhanced eosinophillia observed in PHA lectin treated animals (Meeusen and Balic, 2000), this suggests that there are aspects of the effector mechanism that are being altered and enhanced as a consequence of this lectin treatment. However, since the cell counts and IVDC in the present study come from a single time point at slaughter, further kinetic studies are required to fully evaluate changes in these cell populations. The results of this study suggest that dosing sheep with 2.3 mg PHA/kg LW/day can provide an antiparasitic effect and also enhance immune development with no apparent ill-effects on the animal. The dose rate employed in this study was relatively conservative, due to concern of the toxicity of PHA in sheep and was comparable to the ∼2–3 mg/per kg LW per day maximum dose that can be tolerated by humans without adverse effects (Hunter et al., 2008). In comparison, studies in rats have indicated that concentrations less than 250 mg PHA per kg LW are not harmful and can even act as a potent growth stimulator for the small intestine, binding strongly to the brush border without triggering adverse or toxic responses (Bardocz et al., 1996; Grant, 1999), whilst daily doses of PHA around 500 mg per kg LW or higher impair growth and cause loss of body protein and body lipid, damage of the gut wall and overgrowth of Escherichia coli (Pusztai et al., 1991, 1993; Bardocz et al., 1995; Grant, 1999). There is some evidence of a high tolerance of PHA in sheep as studies have reported adult sheep were able to consume considerable amounts of raw kidney bean without adverse reactions (Osborn et al., 1985; Paduano et al., 1995), however, in most cases the lectin content of the seeds was not reported. Further, Paduano et al. (1995) fed a named cultivar of kidney bean (>400 g per day) to sheep with only limited effects on weight gain. The cultivar used is known to be of high lectin content [∼20 g per kg DM] (Osborn et al., 1985) giving a lectin intake of >260 mg per kg LW per day. This suggests that sheep may, in fact, have quite a high tolerance for kidney bean lectin. Although the maximum dose of PHA that can be safely administered to sheep without adverse effects on either the animal itself or the rumen microbial population is yet to be determined, it is possible higher doses of PHA could have been safely applied to the lambs in the current study, with a potentially greater impact on the nematode infection and/or immune development. Acknowledgments Leyla Ríos de Álvarez is grateful to: “Consejo de Desarrollo Científico y Humanístico de la Universidad Central de Venezuela (CDCH-UCV)” for being the sponsor of her PhD studies and to all Moredun Research Institute-Parasitology Laboratory team and Farm team, for their support and help. References AFRC, 1993. Energy and Protein Requirements of Ruminants. An Advisory Manual Prepared by the Agriculture and Food Research Council Technical Committee on Responses to Nutrients. CAB International, Wallingford, UK. Banwell, J.G., Abramowsky, C.R., Weber, F., Howard, R., Boldt, D.H., 1984. Phytohemagglutinin-induced diarrheal disease. Digest. Dis. Sci. 29, 921–929.

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