International Journal for Parasitology 44 (2014) 697–702

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Vaccination of grazing calves with antigens from the intestinal membranes of Haemonchus contortus: effects against natural challenge with Haemonchus placei and Haemonchus similis C.C. Bassetto a,⇑, M.R.L. Silva a, G.F.J. Newlands b, W.D. Smith b, J. Ratti Júnior c, C.L. Martins c, A.F.T. Amarante a a

UNESP – Universidade Estadual Paulista, Departamento de Parasitologia, Instituto de Biociências, Caixa Postal 510, Botucatu, SP CEP 18.618-000, Brazil Moredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik, EH26 0PZ Edinburgh, UK c UNESP, Departamento de Produção Animal, Faculdade de Medicina Veterinária e Zootecnia, Fazenda Experimental Lageado, Botucatu, SP CEP 18.618-000, Brazil b

a r t i c l e

i n f o

Article history: Received 7 March 2014 Received in revised form 10 April 2014 Accepted 11 April 2014 Available online 21 June 2014 Keywords: Vaccine Cattle Protective antigens Antibodies Gastrointestinal nematodes Haemonchus

a b s t r a c t A vaccine containing integral membrane glycoproteins from the intestine of Haemonchus contortus was evaluated in three groups of eight 5 months old grazing calves, naturally infected by Haemonchus similis, Haemonchus placei and other gastrointestinal nematodes. Vaccinated calves received 5 or 50 lg of the antigen and 1 mg of saponin adjuvant, while the controls received adjuvant alone, initially three times, 3 weeks apart and then four more times at 6 weeks intervals. Three weeks after the last immunisation all of the calves were euthanised for worm counts. Immunisation stimulated high titre antibodies against the vaccine antigens, reduced the egg output of Haemonchus spp. by 85% and the numbers of H. placei and H. similis by 63% and 32%, respectively, compared with control calves. It was concluded that vaccination with intestinal membrane glycoproteins from H. contortus could substantially reduce the transmission of H. placei and H. similis, thus providing protective benefit downstream. This appears to be the first known successful demonstration of a vaccine protective for cattle naturally exposed to infection with any gastrointestinal nematode parasite. Ó 2014 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction Several species of nematodes parasitize the gastrointestinal tract of cattle. In tropical and sub-tropical areas of the world Haemonchus spp. are among the most important. In recent reports from Brazil, the proportion of Haemonchus spp. infective L3s in faecal cultures ranged between 50 and 98% (Borges et al., 2013; Cardoso et al., 2013) with Haemonchus placei and Haemonchus similis usually the predominant species (Amarante et al., 1997). Control of bovine parasitic gastroenteritis currently relies almost entirely on the use of anthelmintic drugs, but drug resistance is increasingly common (Anziani et al., 2004; Gasbarre et al., 2009) including cases in Brazil where Haemonchus spp. are becoming resistant to benzimidazoles, imidazothiazoles and macrocyclic lactones (Soutello et al., 2007; Souza et al., 2008), and

⇑ Corresponding author. Address: Departamento de Parasitologia, Instituto de Biociências, Universidade Estadual Paulista (UNESP), Distrito de Rubião Júnior s/n, Botucatu, São Paulo 18.618-970, Brazil. Tel.: +55 14 3880 0524; fax: +55 14 3815 3744. E-mail address: [email protected] (C.C. Bassetto).

newer compounds such as monepantel and derquantel are not yet registered for bovine use. Certainly, the general problem is very similar to the situation with sheep where anthelmintic resistance is a growing phenomenon on the American continent (reviewed by Torres-Acosta et al., 2012). In addition to the resistance problem, world demand for meat and milk free of chemical residues is increasing, forcing attention towards drug-free approaches to worm control (Fitzpatrick, 2013). One potential option is vaccination. A vaccine for Haemonchus contortus is currently being reviewed by the regulatory authorities in two countries and is expected to be commercially available for Australian sheep farmers during 2014 (Smith et al., 2013a). The vaccine contains a small dose (5 lg) of native glycoproteins isolated from the intestinal cells of the parasite. The same vaccine, except that it contained 50 lg of antigen, has already been shown to protect housed calves artificially challenged with H. contortus or H. placei (Bassetto et al., 2011). The main objectives of the present work were to determine whether the same formulation (50 lg) or a lower dose (5 lg) of antigen could protect grazing calves exposed to natural challenge with mixed species of Haemonchus.

http://dx.doi.org/10.1016/j.ijpara.2014.04.010 0020-7519/Ó 2014 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

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2. Materials and methods 2.1. Animals and management Twenty-four Nellore calves, 12 female and 12 male, born from 29 January to 15 March 2011 were raised grazing on Brachiaria brizantha cv Marandu permanent pasture on a cattle farm of the ‘‘Universidade Estadual Paulista’’ (UNESP), located at 22° 810 S, 48° 400 W, 554 m above sea level near Botucatu, São Paulo State, Brazil. Only cattle had access to this pasture. Cows and their calves were kept in 14 (1.5 ha) paddocks with water and mineral salt available ad libitum. All groups grazed together throughout the trial and when there was insufficient grazing pasture, the animals were moved to a fresh paddock. When the calves were weaned on 15 September 2011, they remained in the same paddocks and the cows were transferred to another farm. The calves were given concentrate from weaning until 15 December 2011 (800 g per calf; Geramilk BezerraÒ, Presence, Brazil) and coccidiostat (decoquinate at 0.5 mg/kg, DeccoxÒ, Alpharma, USA) mixed with the concentrate, until 15 November 2011. All received a clostridial vaccination on 7 July 2011 (Carbun-vet PolivalenteÒ, Biovet, Brazil), a Foot and Mouth Disease vaccination on 10 November 2011 (Aftobov OleosaÒ, Merial, Brazil) and on 6 October 2011 and 22 December 2011 they were treated with cypermethrin (0.5 g/100 kg, CypermilÒ Pour On, Ouro Fino, Brazil) to control ectoparasites (mainly Haematobia irritans). All calves were drenched on the first day of experimental vaccination (28 July 2011, day 0) with albendazole at 10 mg/kg (Endazol 10% CoÒ, Irfa, Brazil) to remove any existing gastrointestinal nematodes.

2.2. Design of the experiment All procedures were performed in accordance with the Animal Welfare Standards and the study was approved by the Animal Research Ethics Committee of the UNESP (protocol 262-CEEA). Calves were allocated to three groups of eight (four females and four males per group) balanced as closely as possible by nematode faecal egg count (FEC). Two groups were vaccinated with either 5 or 50 lg of the antigen formulated with 1 mg of saponin in 1 ml of Tris buffer, pH 7.4 and the third (control) group was vaccinated with adjuvant only. The vaccine antigen containing integral membrane glycoproteins from the intestine of H. contortus was prepared at Moredun Research Institute, UK as described before (Smith et al., 2000) and the vaccine was administered i.m. in the neck. Briefly, Triton X-100 extracts of the parasite membranes were prepared (Smith et al., 1999), diluted fourfold with 10 mM Tris–HCl, 0.5 M NaCl, 0.02% NaN3, pH 7.4 and Ca++ and Mg++ were added to 100 lM and 10 lM, respectively. The solution was pumped (6 ml/h) at 4 °C through ConcanavalinA lectin cross-linked to agarose beads (Vector Laboratories, USA) contained in a column. After thorough washing in the same buffer, the column was eluted with 0.2 M methylmannopyranoside/methylglucopyranoside and the eluate was used as the vaccine antigen. The first immunisation was given on 28 July 2011 (V1 on day 0), when the calves were on average 5 months old and two more injections followed, 3 weeks apart. After weaning on day 49 when the calves were approximately 7 months old, they received four more vaccine boosts, all 6 weeks apart. All calves were euthanised for worm counts on 15 March 2012, 3 weeks after their final vaccination (day 231 after V1), when they were, on average, 13 months old. Blood samples were collected from the jugular vein into a tube containing EDTA (VacutainerÒ, BD, USA) and packed cell volume (PCV) and total plasma protein (TPP) were determined by

microcentrifugation and use of a refractometer, respectively. The plasma was then separated from the blood and frozen (20 °C) for subsequent ELISAs to determine antibody titres against the vaccine antigens (Bassetto et al., 2011). In January 2012, one calf from the 50 lg vaccination group died of causes unrelated to the experimental design of the trial. Its data were excluded from the analysis. 2.3. Faecal examination and worm counts Faecal samples were collected to determine nematode egg counts by a modified McMaster method in which each egg counted represented 50 eggs per gram of faeces (EPG) (Ueno and Gonçalves, 1998). Composite faecal cultures were performed separately for each group and larval identification of faecal cultures followed descriptions by Keith (1953). After the calves were euthanised, the abomasum, small and large intestine were frozen at 20 °C, for between 5 and 10 months. After thawing, each organ was washed separately in saline solution. The abomasal contents were collected and the parasites present in a 20% sub-sample were counted. If fewer than 40 adult Haemonchus were obtained, the remaining 80% of the content was also searched for parasites. In addition, the worms present in a 5% sub-sample of the small intestine and a 10% sub-sample of large intestine contents were collected; all worms attached to the large intestine mucosa were also collected and enumerated. Worms were counted, sexed and classified by their stage of development and species (Vicente et al., 1997; Ueno and Gonçalves, 1998). Female Haemonchus spp. were specified by the morphology of the vulvar flap: they were classified as H. similis if they had a linguiform flap with a long vagina that extended along the vulvar flap and opened near its extremity (Silva et al., 2014). Tail length and tail length as a proportion of body length were also used to distinguish female H. similis from H. placei (Lichtenfels et al., 1994). Twenty male Haemonchus (or all available specimens if fewer than 20 were found) per abomasum sample were chosen at random for examination. Species identification was based on the length of the spicules and barbs, but if these characteristics were doubtful the number of longitudinal ridges in the synlophes were counted (Lichtenfels et al., 1986, 1994). The proportion of males of each species was obtained for each group, which allowed an estimate of the total number of adult male H. similis and H. placei in each animal to be calculated. 2.4. Statistical analyses In the case of body weight, antibody titres, PCV, TPP, general FEC and Haemonchus spp. FEC, the repeated measures analyses of variance was employed in data analyses using the general linear model procedure of SAS release 9.2. Worm burden data were analysed using one-way ANOVA. Significant differences between the group means were determined by Fisher’s least significant difference at a significance level of 5%. Antibody titres, FEC and worm burden were log-transformed (log10(x + 1)) prior to analysis. Arithmetic means ± S.E.M. of the untransformed data are shown throughout. 3. Results 3.1. Antibody response and faecal egg counts Antibody titres close to zero were detected in all calves at the start of the trial and remained low in the control group throughout (Fig. 1A). The vaccinated calves first showed an obvious rise in titre after the second vaccination, irrespective of the dose of antigen,

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and a short-lived peak was observed in both groups after each subsequent booster vaccination, but with no significant difference between them (P > 0.05). Mean titres in both groups remained highly elevated (above 50,000) throughout the trial (Fig. 1A). Most calves were shedding worm eggs at the start of the trial, although very few were Haemonchus spp. (Fig. 1B, C) and Cooperia

(A)

spp. dominated the faecal cultures. As expected, total worm egg counts declined soon after the anthelmintic treatment at the beginning of the trial and remained negligible until mid-September 2011, when a new patent infection by gastrointestinal nematodes had been established. Mean total egg counts in all three groups were then maintained at approximately 100 EPG for the

Vaccine antibodies

Mean antibody titre ± S.E.

400000

5 µg 50 µg

300000

Control Vaccine given 200000

100000

0 0

21

42

63

84 105 126 147 168 189 210 231

Days after V1

Total eggs

(B) 1000

5 µg

Mean EPG ± S.E.

50 µg 750

Control Vaccine given Weaned

500

250

0 0

21

42

63

84 105 126 147 168 189 210 231

Days after V1

Haemonchus spp. eggs

(C) A

S

O

N

D

300

J

F

M 400

300

200

200

100

100

0 0

21

42

63

5 µg

mm rain per month

Mean EPG ± S.E.

400

50 µg Control Vaccine given Rain Months

0 84 105 126 147 168 189 210 231

Days after V1 Fig. 1. Kinetics of total (B) and Haemonchus-specific faecal egg counts (eggs per gram of faeces, EPG, (C)) in relation to rainfall and vaccine antibody titres (A) of calves immunised with 5 or 50 lg of Haemonchus contortus gut membrane antigen or vaccinated with adjuvant only (control). VI, first immunisation – day 0. A, August; S, September; O, October; N, November; D, December; J, January; F, February; M, March.

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C.C. Bassetto et al. / International Journal for Parasitology 44 (2014) 697–702

Haemonchus spp. eggs from day 168 to 231

Mean EPG ± S.E.

300

200

100

variable, such that statistical significance was only attained with male adult H. placei (Table 1). Besides Haemonchus, five other nematode genera were recovered from the calves. Cooperia punctata was the most numerous species, followed by smaller numbers of Oesophagostomum radiatum, Trichostrongylus axei, Trichuris discolour and a few specimens each of Cooperia spatulata, Trichostrongylus longispicularis and Agriostomum vryburgi (Table 2). There was no indication that vaccination had any effect on numbers of any of the non-Haemonchus spp. 3.3. PCV, TPP concentrations and live weight gains

0 5 µg

50 µg Group

Control

Fig. 2. Mean numbers of Haemonchus eggs shed by individual calves in the final 9 weeks of the trial with calves immunised with 5 or 50 lg of Haemonchus contortus gut membrane antigen or vaccinated with adjuvant only (control).

next 6 weeks, but increased with the onset of rain in October, 2011 to fluctuate between 300 to 700 EPG in the control group for the rest of the trial. Mean values in both vaccinated groups were nearly always lower but not significantly so (P > 0.05). Haemonchus-specific mean egg counts remained below 40 EPG in all three groups until January, 2012 when they rose to some 200 EPG in the control group only (Fig. 1B). Mean control group Haemonchus spp. eggs then fluctuated around 100 EPG until the end of the trial but the counts in the vaccinated calves were significantly lower (P < 0.01) during this period from January until the end of the trial (Figs. 1C, 2). 3.2. Worm counts The mean number of adult male or female Haemonchus spp. recovered from the vaccinated calves was always lower than the equivalent control values. However, individual burdens were quite

Almost all PCV measurements were in the normal 30% to 40% range (results not shown). There was no indication of lower values in the Control group during the last 2 months of the trial when Haemonchus egg counts were elevated. Nor was the presence of more than 4,000 Haemonchus in three calves at post-mortem associated with an obvious drop in PCV. Plasma protein concentrations ranged from 5.5 to 7.0 g/dl in all three groups (results not shown). No correlation was observed between the concentration in the final blood samples collected and the total worm burden. Each calf gained an average of 68 kg (S.D. ± 15.5) during the trial. At no stage were any significantly different growth rates observed between the groups, nor was there any correlation between final worm burden and weight gain during the last 8 weeks of the trial (results not shown). 4. Discussion The overall aim of this trial was to assess the ability of two different doses of a native H. contortus antigen vaccine to protect calves against a natural field challenge with Haemonchus spp. An experimental vaccine containing 50 lg of the same antigen per dose had been shown previously to protect housed calves against an artificial bolus challenge of H. placei (Bassetto et al., 2011).

Table 1 Haemonchus similis and Haemonchus placei worm burdens of calves immunised with 5 or 50 lg of Haemonchus contortus gut membrane antigen or vaccinated with adjuvant only (control). Stage of development

Juveniles (L4 + Early L5) H. similis adult female H. similis adult male H. placei adult female H. placei adult male

Group

P value

5 lg

50 lg

Control

408a (14–2,360) 318a (0–1,176) 343a (1–1,365) 76a (0–459) 75a (0–300)

564a 495a 546a 50a,b 89a,b

467a (30–1,135) 623a (205–1,135) 603a (160–1,356) 132b (25–325) 246b (65–554)

(21–1,890) (18–1,085) (60–1,286) (0–178) (10–209)

>0.51 >0.15 >0.15

Vaccination of grazing calves with antigens from the intestinal membranes of Haemonchus contortus: effects against natural challenge with Haemonchus placei and Haemonchus similis.

A vaccine containing integral membrane glycoproteins from the intestine of Haemonchus contortus was evaluated in three groups of eight 5 months old gr...
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