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Resistance of Santa Ines and crossbred ewes to naturally acquired gastrointestinal nematode infections
Veterinary Parasitology 165 (2009) 273–280
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Resistance of Santa Ines and crossbred ewes to naturally acquired gastrointestinal nematode infections A.F.T. Amarante a,*, I. Susin b, R.A. Rocha a, M.B. Silva a, C.Q. Mendes b, A.V. Pires b a b
UNESP - Universidade Estadual Paulista, Departamento de Parasitologia, Instituto de Biocieˆncias, Caixa Postal 510, Botucatu, SP, CEP 18618-000, Brazil USP – Universidade de Sa˜o Paulo, Departamento de Zootecnia – ESALQ, Piracicaba, SP, CEP 13418-900, Brazil
A R T I C L E I N F O
A B S T R A C T
Article history: Received 7 April 2009 Received in revised form 17 June 2009 Accepted 7 July 2009
This trial was carried out in Piracicaba, Sa˜o Paulo State, Brazil, to comparatively evaluate the degree of resistance to naturally acquired gastrointestinal nematode infections in sheep of the following genetic groups: purebred Santa Ines (SI), SI crossbred with Dorper (DO SI), Ile de France (IF SI), Suffolk (SU SI), and Texel (TE SI). Fifteen ewes from each group were raised indoors until 12 months of age. At this age, they were moved to pasture that was naturally contaminated by nematode infective larvae and were evaluated from December to May, 2007. Rainfall ranged from 267 mm in January to 37 mm in April. Maximum and minimum mean temperatures ranged from 32.5 8C to 19.0 8C in March and from 25.9 8C to 12.8 8C in May. There was an increase in the mean number of eggs per gram of feces (EPG) after animals were placed on pasture with significant difference between the SI (80 EPG) and IF SI (347 EPG) groups in January; and the DO SI (386 EPG) and TE SI (258 EPG) groups in May. The highest mean fecal egg count (FEC), 2073 EPG, was recorded for the TE SI group in February. All groups showed a progressive reduction in body weight throughout the experiment of 12.0% (TE SI) to 15.9% (SU SI). In general, the animals with the highest FEC presented the lowest packed cell volumes (PCV); the highest correlation coefficient between FEC PCV occurred in the SU SI sheep in January (r = 0.70; P < 0.01). Similarly, there was an inverse relationship between FEC and blood eosinophil values, with the highest correlation coefficient in the TE SI sheep in February (r = 0.64; P < 0.05). Immunoglobulin G (IgG) levels against Haemonchus contortus antigens increased in all groups as a result of the exposure to parasites and remained relatively constant until the end of the study, with the exceptions of SU SI and TE SI, which showed a rise in IgG levels during the last sampling that coincided with a reduction in mean FEC. In conclusion, crossbreeding Santa Ines sheep with any of the breeds evaluated can result in a production increase and the maintenance of a satisfactory degree of infection resistance, especially against H. contortus and Trichostrongylus colubriformis, the major nematodes detected in this flock. ß 2009 Elsevier B.V. All rights reserved.
Keywords: Haemonchus Trichostrongylus Genetic resistance Immune response Immunoglobulins IgG
1. Introduction Parasitic gastroenteritis is a major health problem for the sheep industry worldwide, causing great economical losses due to reduced productivity, mortality of animals,
* Corresponding author. Tel.: +55 14 3811 6239; fax: +55 14 3815 3744. E-mail address: [email protected] (A.F.T. Amarante). 0304-4017/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2009.07.009
and expenses with anthelmintics and labor. In addition, the frequent use of anthelmintics for the prophylaxis of gastrointestinal nematode (GIN) infections has led to the dissemination of populations of resistant parasites (Getachew et al., 2007). Alternative control measures include breeding strategies with the selection or the use of sheep breeds with resistance to GIN. This strategy has demonstrated sustainability, as strong evidence suggests that important sheep
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A.F.T. Amarante et al. / Veterinary Parasitology 165 (2009) 273–280
nematodes do not adapt to long-term exposure to sheep that are genetically resistant to GIN infections (Kemper et al., 2009). Some breeds of sheep demonstrate genetic resistance against Haemonchus contortus, the major parasite in tropical and sub-tropical areas; such breeds include the Florida Native (Amarante et al., 1999a) and Gulf Coast Native (Bahirathan et al., 1996; Li et al., 2001) sheep, raised in the south of the United States; St. Croix and Barbados Blackbelly (Courtney et al., 1984; Gamble and Zajac, 1992; Gruner et al., 2003), originating from the Caribbean islands; and the Red Maasai sheep from Africa (Mugambi et al., 1997). In Brazil, Santa Ines hair sheep demonstrate a greater resistance to GIN, when compared with some sheep breeds of European origin (Amarante et al., 2004; Rocha et al., 2004; Bricarello et al., 2005; Costa et al., 2007). These differences in breeds are usually the result of emphasis on different traits in selection, or of natural selection in breeds originating from different geographical locations. As such, problems with GIN infections in the sheep industry could be reduced by the use of resistant breeds; however, these resistant breeds can present inferior productivity when compared with other breeds selected for higher weight gain and meat quality. For example, the Ile de France is a breed notable for weight gain and carcass quality; however, its performance in tropical environments may not match that of tropical breeds adapted to the area, such as the Santa Ines hair sheep. Therefore, the farmer has to choose between a less productive breed that is well-adapted to the local environmental conditions and resistant to parasites or a highly productive breed that is not well-adapted to the climate and is more susceptible to parasites that occur in the tropics. A third option to solve such a problem is the crossbreeding of a susceptible breed, of elevated productivity, with a resistant, but less productive breed. The variation between breeds presents opportunities to combine and improve the characteristics of two different breeds in order to enhance productivity. In addition, crossbred progeny usually show heterosis in performance. Thus, crossbreeding allows the opportunity to obtain progress in a generation that would normally need several generations of selection to be reached (Van Vleck et al., 1987). At present, the Santa Ines sheep is predominant in most of the Brazilian territory due to its higher level of resistance to GIN (Amarante et al., 2004; Rocha et al., 2004; Bricarello et al., 2005; Costa et al., 2007). This trial was carried out to evaluate the degree of resistance of female sheep originating from the crossbreeding of Santa Ines ewes with Ile de France, Suffolk, Texel and Dorper sires, all breeds with a high potential for growth and meat production. 2. Materials and methods 2.1. Experimental area and animal management This study was carried out on an experimental farm located at the University of Sa˜o Paulo in Piracicaba, Brazil. The experimental area comprised 1 ha of grass pasture (Cynodon spp.) divided into a six-paddock rotational grazing system.
The average monthly relative humidity was always higher than 84% throughout the study. As usual for the region, rainfall was abundant in December, January and February (252 mm, 267 mm and 242 mm, respectively), with a total of 761 mm. In March (81 mm), April (37 mm) and May (58 mm), there was a decrease in precipitation. Maximum and minimum mean temperatures ranged from 32.5 8C to 19.0 8C in March 2007 and from 25.9 8C to 12.8 8C in May (ESALQ, 2009). Each experimental genotype group was composed of 15 young ewes, as follows: purebred Santa Ines (SI); crossbred Dorper Santa Ines (DO SI); crossbred Ile de France Santa Ines (IF SI); crossbred Suffolk Santa Ines (SU SI); and crossbred Texel Santa Ines (TE SI). The ewe lambs used in this trial were obtained by crossing 300 Santa Ines females with five sires of each of the following breeds: Santa Ines, Dorper, Texel, Suffolk and Ile de France. Each sire was exposed to the same number of females to assure genetic variability inside each experimental group. The birth date, the birth weight and the weight at weaning (60 days of age) were recorded, as well as whether the lambs were single or twins. This procedure allowed homogeneity for each genetic group. Animals were managed, according to routine procedures, at the sheep experimental farm. During late pregnancy, ewes were housed in pens with a concrete floor, where they were kept with their lambs until weaning. Ewes were vaccinated against clostridial disease (Sintoxan Polivalente1, Merial S.A., Uruguay), salmonelloses and pasteurelloses (Laborato´rio Prado S.A., Brazil) in the final third of the gestation. Lambs were vaccinated 15 days before weaning. Suckling lambs had free access to a concentrate with 18% crude protein in a creep feeding system. After weaning, lambs received a diet containing 16% crude protein, and consisting of hay (10%) and concentrate (90%). The concentrate was formulated from soybean meal, ground corn and mineral supplement. In order to prevent coccidiosis, monensin (25 ppm; Rumensin1, Elanco, Australia) was added to the diet. From 6 to 12 months of age, animals were fed on a higher roughage diet to avoid excessive fat deposition. Until the beginning of the trial, all animals were kept housed with free access to drinking water. At 12 months of age, the ewe lambs were released on pasture on December 19th, 2006, where they grazed, in a rotational system, as a single flock, in six paddocks until the end of the study. Pastures were expected to be naturally contaminated with infective larvae of GIN. In each paddock, ewe lambs had free access to water and mineral salt. The animals were weighed monthly and individual samples of blood and feces were taken. To avoid deaths, ewes were individually treated with levamisole (7.5 mg/ kg, Ripercol1, Fort Dodge, Brazil) whenever their fecal egg counts (FEC) were higher than 4000 eggs per gram (EPG) or their packed cell volume (PCV) was lower than 20% (Amarante et al., 1999b). During the last two months of the study, two tracer lambs, free of infections of GIN, were placed together with the experimental animals. At the end of the study, these tracer lambs were sacrificed in order to identify species of helminths present in the flock.
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2.2. Parasitology Fecal samples were taken directly from the rectum of the animals for fecal egg count (FEC) determination using a modified McMaster technique (Ueno and Gonc¸alves, 1998). Each egg counted represented 100 eggs/g. Composite fecal cultures were performed separately for each group of sheep for production of third stage larvae, which were identified according to the descriptions of Keith (1953). At necropsy of the two tracer lambs, the abomasum and the small and large intestines were removed, opened and the contents placed in graduated buckets. A 10% aliquot was preserved in 4% formalin for subsequent enumeration of the helminths and identification to species level of the adult male nematodes, based on the morphology of their spicules (Ueno and Gonc¸alves, 1998). Pasture samples were also collected on the day of fecal and blood collection to assess the number of infective GIN larvae per kilogram of dry matter (L3/kg DM) from two of the paddocks grazed by the experimental animals. For pasture sampling, the grass collector followed a W-shaped track in each paddock, collecting samples every four steps (approximately 3.5 m). Grass was cut as close to the ground as possible and the samples were processed in the laboratory, as described by Amarante and Barbosa (1995).
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protease inhibitor solution (Complete Mini Solution1, Roche, USA) using an Ultra Turrax1 (Ika, Germany). The extract was centrifuged (15,000 g) at 4 8C for 20 min and the supernatant containing the adult-soluble-antigen extract was collected. Protein concentrations of L3 and adult antigens were determined using a kit (Protal me´todo colorime´trico1, Laborlab, Brazil) and absorbance was read at 562 nm. The antigen extracts were stored in aliquots at 80 8C until further use. 2.6. Parasite-specific serum IgG
Blood samples with EDTA and without anti-coagulant were collected by jugular veni-puncture. Packed cell volume (PCV) was determined by the micro-hematocrit method. Serum samples were stored at 20 8C until use for immunoglobulin measurements. The eosinophil counts in peripheral blood were made in a Neubauer’s chamber after staining with Carpentier’s solution (Dawkins et al., 1989); counts were expressed as the number of cells per ml of blood.
Polystyrene micro-titer plates (Nunc, USA) were coated with 100 ml of adult or L3 H. contortus (2 mg/ml) antigens diluted in carbonate–bicarbonate buffer (pH 9.6); plates were incubated overnight at 25 8C. All subsequent incubations were carried out for 1 h at 37 8C using, in each well, a total of 100 ml of reagents. Between each step, plates were washed three times with ultra pure water (EASYpure II UV, Barnstead, USA) containing 0.05% Tween 20. After coating, blocking was carried out with 0.1% Gelatin (Amresco, USA) and 0.05% Tween 20 (Amresco, USA) in PBS (PBS–GT). Serum samples were diluted in PBS– GT (1:1000) and applied in duplicate. Plates were then incubated with rabbit polyclonal antibody to sheep IgG (Abcam Inc. UK; 1:2000 in PBS–GT) followed by polyclonal goat anti-rabbit immunoglobulins linked to alkaline phosphatase (Dako, Denmark; 1:2000 in PBS–GT). Finally, substrate solution (pnitrophenyl phosphate disodium salt, PNPP, Pierce, USA) was added to each well and the enzymatic reaction was allowed to proceed at room temperature for 30 min and plates were immediately read using an automated ELISA reader (Biotrak II, AmershamBiosciences, UK) at 405 nm. The standard positive serum was obtained from a sheep repeatedly infected with H. contortus. Results were expressed as the percentage of the optical density value of the positive-reference serum (Kanobana et al., 2002).
2.4. Enzyme-linked immunosorbent assay
2.7. Statistical analysis
Immunoglobulin G (IgG) levels against larval and adult H. contortus antigens were measured in serum samples.
Significant differences between groups for FEC, PCV, body weight, eosinophils and IgG levels were assessed by oneway analysis of variance using the statistical software, Minitab 11.21 (Minitab Inc., USA). Group means were compared using the least significant difference at P < 0.05 (Ott, 1992). FEC data were transformed using log10(x + 1) prior to analysis. The figures in the results are expressed as arithmetic means (standard error of the mean). Spearman’s correlation coefficient between variables was assessed using the same software.
2.3. Hematology
2.5. Larval and adult H. contortus antigens Infective H. contortus third stage larvae (L3) were obtained from fecal cultures of monospecifically infected donor sheep. The same donor sheep was sacrificed and adult worms were recovered from the abomasum and washed several times in PBS. H. contortus L3 were exsheathed, under microscopic monitoring, with 0.1% sodium hypochlorite solution and subsequently washed in PBS. Larvae were fragmented at 4 8C in PBS supplemented with protease inhibitor solution (Complete Mini Solution, Roche, USA) using an ultrasonic processor (VibraCell–Sonics & Materials Inc., USA). The homogenized content was centrifuged at 15,000 g, at 4 8C, for 20 min and the supernatant containing the L3-solubleantigen extract was collected. Adult H. contortus worms were fragmented at 4 8C in PBS supplemented with 2 ml of
3. Results At the beginning of the trial, some sheep were passing nematode eggs in feces, but in a small number. The maximum FEC was 900 EPG in one SU SI ewe (Table 1). These findings demonstrated that, while housed, young ewes were exposed to an apparently low level of infective larval (L3) contamination. After being introduced to their grazing paddocks, the ewes started to present a higher
TE SI (n = 15)
93.3 (0; 500), 0.79ab 0.30 1257.0 (0; 12,600), 1.64ab 0.41 2073.0 (0; 14,700), 1.83a 0.43 260.0 (0; 1220), 1.34a 0.36 980.0 (0; 4400), 2.19a 0.32 258. 0 (0; 1200), 1.15b 0.41
SU SI (n = 15)
246.7 (0; 900), 1.90a 0.26 1007.0 (0; 4500), 1.62ab 0.41 720.0 (0; 2900), 2.21a 0.31 253.3 (0; 1300), 1.72a 0.29 1485.0 (0; 14,200), 1.57a 0.44 342.0 (0; 1100), 1.87ab 0.34 6.7 (0; 100), 0.13b 0.13 347.0 (0; 2200), 1.95b 0.28 1227.0 (0; 7500), 2.08a 0.36 146.7 (0; 400), 1.54a 0.29 1086.0 (0; 6000), 2.17a 0.35 527.0 (0; 4500), 1.64ab 0.34 6.7 (0; 100), 0.13b 0.13 146.7 (0; 600), 1.26ab 0.32 1453.0 (0; 9500), 2.26a 0.33 333.0 (0; 1500), 1.93a 0.28 940.0 (0; 5500), 1.84a 0.37 386.0 (0; 1600), 2.19a 0.26
IF SI (n = 15) DO SI (n = 15) SI (n = 15)
146.7 (0; 600), 1.14a 0.26 80.0 (0; 400), 0.77a 0.29 320.0 (0; 1800), 1.65a 0.33 266.7 (0; 1500), 1.89a 0.27 240.0 (0; 1500), 1.70a 0.29 814.0 (0; 9300), 1.78ab 0.34
Data
19/December 16/January 13/February 15/March 11/April 10/May
Table 1 Fecal egg counts of the Santa Ines (SI), Dorper Santa Ines (DO SI), Ile de France Santa Ines (IF SI), Suffolk Santa Ines (SU SI) and Texel Santa Ines (TE SI) young ewes during natural infection by gastrointestinal nematodes.
Results are arithmetic mean with minimum and maximum values in parenthesis. Means of log transformed values (standard error) followed by different superscripts in the same line are significantly different (P < 0.05).
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Fig. 1. Means of packed cell volume (A) and blood eosinophils (B) of the Santa Ines (SI) purebred sheep and Dorper Santa Ines (DO SI), Ile de France Santa Ines (IF SI), Suffolk Santa Ines (SU SI) and Texel Santa Ines (TE SI) crossbred sheep during natural infection by gastrointestinal nematodes. Bars represent standard error of the mean.
degree of infection. Significant difference among group means occurred between SI (80 EPG) and IF SI (347 EPG) groups in January, and between DO SI (386 EPG) and TE SI (258 EPG) in May. The highest FEC mean value (2073 EPG) was recorded in TE SI sheep in February (Table 1). The PCV averages showed a progressive reduction throughout the experiment (Fig. 1). Significant differences between group means were registered only at the beginning of the study: the average of the animals SI (33.6 0.52%) was lower than the average of the DO SI (36.5 1.04%) and IF SI (36.3 0.59%) in December, and the averages of the SU SI (29.9 1.52%) and TE SI (30.2 1.43%) groups were significantly lower than the averages of the SI (33.6 0.65%), DO SI (34.6 1.06%) and IF SI (34.3 0.61%) groups in January. In order to avoid cases of mortality, the animals were treated individually with anthelmintics when they presented FEC higher than 4000 EPG or PCV values lower than 20%. There was no case of any animal needing more than one treatment, with the exception of one SU SI ewe. The animals treated were as follows: one SI ewe (in May), two DO SI (both in February), three IF SI (February, April and May), two SU SI (one in January and other twice, in January and in April) and five TE SI (one in January, three in February and one in April). The percentage reduction in FEC was greater than 93% at seven days after the administration of levamisole. The lowest eosinophil averages occurred at the beginning of the study. In the following months, average counts increased in all groups and were usually higher than 1000 cells/ml (Fig. 1). During the last month of the study, the averages of the SI and TE SI groups were significantly lower than those of the other groups.
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Table 2 Mean levels expressed as a percent of standard serum (standard error) of IgG against antigens of third stage larvae (L3) and adults of Haemonchus contortus in the serum of the Santa Ines (SI), Dorper Santa Ines (DO SI), Ile de France Santa Ines (IF SI), Suffolk Santa Ines (SU SI) and Texel Santa Ines (TE SI) young ewes during natural infections by gastrointestinal nematodes. Date
Antigen
DO SI (n = 15)
IF SI (n = 15)
SU SI (n = 15)
TE SI (n = 15)
19/December
L3 Adult
SI (n = 15) 54.7a 3.86 33.5a 2.57
64.2a 5.74 48.3a 6.37
49.0a 3.88 28.7b 3.65
92.3b 6.15 87.2c 5.54
82.5b 6.79 79.0c 5.77
16/January
L3 Adult
112.0a 3.75 101.9a 4.33
101.3ab 5.3 100.9a 5.46
85.6b 5.76 88.5a 6.05
99.0ab 7.35 110.3a 7.51
91.2b 6.39 93.8a 6.94
13/February
L3 Adult
104.9a 4.46 97.2ac 3.92
99.6a 6.14 87.8a 5.45
94.2a 6.09 86.1a 5.22
102.4a 5.45 112.8b 3.80
96.6a 5.92 103.3bc 4.16
15/March
L3 Adult
111.9ac 2.58 111.4a 3.01
109.1ac 4.05 103.7a 4.74
94.8 b 3.76 94.8a 6.15
115.2c 3.55 108.6a 3.97
102.8ab 5.64 98.3a 4.43
11/April
L3 Adult
119.2a 3.94 117.8a 4.24
116.6a 3.74 120.9a 2.99
101.7b 3.42 104.9b 6.63
117.3a 4.45 119.6a 4.88
100.2b 4.77 104.1b 3.57
10/May
L3 Adult
105.6a 1.55 104.1a 2.59
104.1ab 2.43 102.7a 2.91
98.2b 1.87 103.8a 2.16
149.4c 3.13 147.6b 3.89
138.6d 2.39 133.9c 3.70
Means followed by different superscripts in the same line are significantly different (P < 0.05).
Analysis of immunoglobulin concentrations in the serum indicated that IgG means against H. contortus antigens were considerably higher in the SU SI and in the TE SI groups at the beginning and at the end of the study (Table 2). The lowest means were detected in the first sampling, reflecting the low exposure of the animals to GIN, while they were housed. With regard to the groups, the lowest means of IgG against L3 were recorded in the IF SI, except on April 11th. The same occurred for the adult antigens: IF SI presented the lowest means, except for the last sampling (May 10). Correlation coefficients between IgG against L3 and IgG against adult antigens were high in all samplings (r from 0.764 to 0.922; P < 0.001). With regard to body weight, the SI animals were the lightest, while the DO SI and TE SI groups presented intermediate body weight averages and the SU SI and IF SI groups were the heaviest (Table 3). There was a progressive reduction in average weight until the end of the experiment. At the end of the experiment, the animals had lost, on average, from 12.5% (TE SI) to 15.9% (SU SI) of their body weight, compared with their initial weight. These two group mean values were significantly different. Haemonchus spp. infective larvae were predominant in fecal cultures with percentages ranging from 42% to 94% in the DO SI group; 64% to 92% in IF SI; 45% to 92% in SI; 34% to 91% in SU SI; and 40% to 92% in TE SI. The
highest Trichostrongylus spp. percentages (51%) occurred in the SI Group on March 15th. Cooperia spp. and Oesophagostomum spp. infective larvae were detected, although in low percentages; the highest values were, respectively, observed in cultures from the DO SI group in March (15%) and in May (24%). Eggs of Strongyloides spp. were frequently detected in fecal samples collected from February to May, but in low numbers. The highest mean Strongyloides spp. FEC was recorded in DO SI ewes in May (235.7 EPG). Besides nematode eggs, oocysts of Eimeria spp. and eggs of Moniezia spp. were also found in some of the fecal examinations. Haemonchus spp. and Trichostrongylus spp. third stage larvae were found in pasture (Fig. 2). The peak in grass contamination occurred in April, when 703.5 Haemonchus spp. L3/kg DM and 309.1 Trichostrongylus spp. L3/kg DM were recorded. Three nematode species were recovered from tracer animals, with the following mean number of worms: 1200 H. contortus, 3121 Trichostrongylus colubriformis and 240 Cooperia curticei. In general, the animals from each group with the highest FEC presented the lowest PCV values and blood eosinophils, corresponding to negative correlation coefficients (Table 4). The highest correlation coefficients between FEC IgG against adult H. contortus antigens
Table 3 Mean (standard error) body weight (kg) of the Santa Ines (SI), Dorper Santa Ines (DO SI), Ile de France Santa Ines (IF SI), Suffolk Santa Ines (SU SI) and Texel Santa Ines (TE SI) young ewes during natural infection by gastrointestinal nematodes. Data
SI (n = 15)
DO SI (n = 15)
IF SI (n = 15)
SU SI (n = 15)
TE SI (n = 15)
19/December 16/January 13/February 15/March 11/April 10/May Weight reduction (kg)a Weight reduction (%)a
50.1a 1.18 45.2a 1.21 45.5a 1.27 47.1a 1.24 46.1a 1.31 43.6a 1.36 6.5a 0.65 13.2ab 1.38%
56.5b 1.26 52.3b 1.13 52.0b 1.11 51.8b 1.15 51.2b 1.24 48.4b 1.35 8.1a 0.80 14.4ab 1.36%
61.6c 1.58 57.5c 1.64 57.3c 1.50 56.0cd 1.51 56.7c 1.45 53.8c 1.26 7.8a 0.65 12.5ab 0.90%
65.4c 1.98 59.1c 1.78 57.7c 1.61 57.4 d 1.47 57.3c 1.51 54.8c 1.36 10.6b 1.03 15.9a 1.21%
56.5b 1.39 52.0b 1.26 51.8b 1.37 53.1bc 1.07 52.4b 1.22 49.6b 1.07 6.9a 0.75 12.0b 1.20%
Means followed by different superscripts in the same line are significantly different (P < 0.05). a Weight reduction = last body weight initial body weight.
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4. Discussion
Fig. 2. Mean Haemonchus spp. and Trichostrongylus spp. third stage larvae by kilogram of herbage dry matter (L3/kg DM) recovered from pastures grazed by the experimental sheep.
were recorded in TE SI group in the last two months of the trial (respectively, r = 0.77 and 0.78; P < 0.01). Correlation coefficients between FEC IgG against L3 antigens were usually lower, the maximum value (r = 0.57; P < 0.05) was found in the SI group in April. All correlation coefficients between FEC and IgG were negative and several were statistically significant in April (Table 4). Correlation coefficients between FEC and body weight were low (Table 4).
Table 4 Correlation coefficients between fecal egg count (FEC) and the following variables: packed cell volume (PCV), number of eosinophils in blood (Eos), body weight (BW), IgG against L3 (IgG-L3) and adult (IgG-adult) H. contortus antigens of the Santa Ines (SI), Dorper Santa Ines (DO SI), Ile de France Santa Ines (IF SI) and Suffolk Santa Ines (SU SI), naturally infected with gastrointestinal nematodes. Date
Group
16/January
SI SI DO SI IF SI SU SI TE
0.31 0.49 0.35 0.70** 0.66*
0.31 0.11 0.43 0.11 0.09
13/February
SI SI DO SI IF SI SU SI TE
0.17 0.58* 0.50 0.38 0.65**
15/March
SI SI DO SI IF SI SU SI TE
11/April
10/May
* **
P < 0.05. P < 0.01.
FEC PCV
FEC BW
FEC Eos
FEC IgG-L3
FEC IgG-Ad
0.58* 0.08 0.42 0.62* 0.39
0.04 0.44 0.41 0.04 0.54*
0.13 0.35 0.45 0.13 0.66**
0.13 0.08 0.06 0.56* 0.18
0.51 0.16 0.16 0.09 0.64*
0.22 0.11 0.55* 0.12 0.05
0.23 0.35 0.44 0.08 0.02
0.45 0.26 0.25 0.31 0.26
0.26 0.20 0.41 0.10 0.41
0.45 0.07 0.55 0.10 0.24
0.12 0.30 0.17 0.27 0.27
0.35 0.01 0.09 0.07 0.63*
SI SI DO SI IF SI SU SI TE
0.01 0.38 0.51 0.32 0.25
0.22 0.02 0.25 0.21 0.28
0.21 0.63* 0.12 0.39 0.50
0.57* 0.56* 0.26 0.40 0.52*
0.36 0.25 0.28 0.72** 0.77**
SI SI DO SI IF SI SU SI TE
0.42 0.02 0.16 0.14 0.53
0.08 0.38 0.10 0.48 0.03
0.33 0.41 0.16 0.57 0.49
0.15 0.13 0.32 0.33 0.33
0.08 0.20 0.07 0.21 0.78**
All animals, raised indoors since birth, receiving a balanced diet and were exposed to a low level of GIN infection, were in excellent conditions at the beginning of the study. After the first sampling, the animals were kept exclusively on pasture, and were exposed to a higher level of parasitic infection, as well to the environmental conditions, characterized in the region, by a summer with high rainfall and temperature. As a consequence of the infections, the change in food supply and, possibly, as a result of exposure to the weather conditions, the animals lost weight, associated with a decrease in PCV and increase in FEC. The changes in these variables were possibly caused, in part, by GIN infections, especially H. contortus, a blood sucking parasite and the major species found in the flock, followed by T. colubriformis. These are the major sheep nematodes in the region (Amarante et al., 2004; Rocha et al., 2008). An increase in numbers of infective larvae on pasture occurred in the autumn, associated with a reduction in rainfalls. These findings are also in agreement with another study carried out in the region (Amarante et al., 2004) and suggest that the heavy summer rains can remove larvae from the herbage. The acquisition and maintenance of immunity to gastrointestinal parasites in sheep is a nutritionally costly process that induces a relatively short-term diversion of nutrients from productive to immunological tissues (reviewed by Greer, 2008). The costs for the development of immune protection against the GIN parasites might have been another reason for the reduction in body weight of the animals in the present study. The resistance was not uniform among animals, even in the same group. Of the 15 TE SI ewes, five needed one anthelmintic treatment due to the presentation of high FEC and/or low PCV, while the other animals of the same group (10 ewes) remained healthy. In the other groups, a smaller number of animals needed anthelmintic treatment: three IF SI, two SU SI, two DO SI and only one SI. After being treated, animals recovered and developed a degree of resistance that was apparently effective for avoiding the establishment of a new heavy re-infection. The exception was one SU SI ewe that was treated twice, in January and in April. Several studies have shown that the Santa Ines hair sheep are more resistant to GIN infections than wool sheep such as Suffolk and Ile de France breeds (Amarante et al., 2004; Rocha et al., 2004; Bricarello et al., 2005; Costa et al., 2007). In the present study, with few exceptions, the F1 animals produced by crossing both susceptible breeds (Suffolk or Ile de France) with Santa Ines animals, resulted in hybrids with a degree of resistance to GIN infections similar to that of the parental resistant breed. The same occurred with SI DO crossbred animals, and to a lesser extent in the SI TE group. The results of other studies carried out with animals produced by the crossing of resistant with susceptible breeds have demonstrated that the degree of resistance of the hybrids can vary as a function of the breeds evaluated, the age of the animals and whether the evaluations were from natural or artificial infections. As such, different
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results were reported in sheep produced by crossing Florida Native (resistant) and Rambouillet (susceptible) breeds. In the evaluations with adult ewes, the F1s showed an elevated degree of resistance to natural infections, similar to that shown by the resistant parental Florida Native breed (Amarante et al., 1999a). Conversely, in lambs artificially infected with H. contortus, the F1s presented a worm burden that was similar to the susceptible Rambouillet lambs (Amarante et al., 1999b). Similarly, in an evaluation of the phenotypic performance of a Red Maasai and Dorper double backcross resource population, Mugambi et al. (2005) observed that the Red Maasai do not respond to artificial challenge with H. contortus in the way that they do to natural infection, suggesting that phenotyping of Red Maasai and its cross-lambs, for purposes of selecting those that are resistant or susceptible, is best done under natural challenge. As observed in the present study, crossbred animals of the Suffolk and Dorper breeds also showed a relatively high degree of resistance in studies carried out in the USA. Heterosis analysis showed that FEC, PCV, nematode count and weight gain of the crossbred Suffolk and Gulf Coast Native lambs favored the Native group (Li et al., 2001); and the relative resistance of mature Dorper crossbred (Dorper Romanov or Dorper Romanov St. Croix) ewes was comparable to that of Katahdin and St. Croix ewes and superior to that of Hampshire ewes under natural or artificial infection (Burke and Miller, 2002). As a consequence of exposure to nematode larvae on pasture, an increase in IgG levels and eosinophilia was evident in all groups. The development of eosinophilia is a characteristic feature of helminth infection and there is strong evidence of an important role of eosinophils in the defense against infective larvae of GIN (Balic et al., 2006; Terefe et al., 2007). One month of exposure to parasites on pasture resulted in an elevation of IgG levels, which remained relatively constant until the end of the study, with the exception of the SU SI and TE SI groups, which showed a rise in IgG in the last sampling, which coincided with a reduction in mean FEC. Analysis of the coefficients of correlation demonstrated that the levels of antibodies had a greater influence on FEC of the TE SI animals, in comparison with other groups. During the last three months of the study, the TE SI animals showed significant negative correlation coefficients between FEC and the levels of IgG against adult antigens of H. contortus, i.e., animals that produced more antibodies were more resistant to GIN parasites. The TE SI group also had the highest number of animals that needed individual anthelminthic treatment (5 of 15 ewes). These results indicate that the animals with the Texel background had a higher genetic variability, which is in agreement with the description by Walling et al. (2004), who found increased heterozygosity and allele variants in Texel, compared to Suffolk sheep. Comparatively, crossbred young ewes presented a similar resistance to GIN infections to that of their purebred Santa Ines counterparts and were heavier. Therefore, all breeds tested can be used with good results in crossing programs with Santa Ines sheep. Other studies
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are necessary to evaluate the performance of crossbred ewes used in a ‘‘three-cross’’ system. In conclusion, crossbreeding of Santa Ines sheep with breeds with a high potential of production provides promise as a measure to increase sheep production, with the maintenance of a satisfactory degree of resistance to gastrointestinal nematode parasite infection. Acknowledgements This study was funded by Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FAPESP). A.F.T. Amarante, I. Susin and A.V. Pires received support from CNPq and R.A. Rocha, C.Q. Mendes and M.B. Silva from FAPESP. References Amarante, A.F.T., Barbosa, M.A., 1995. Seasonal variations in populations of infective larvae on pasture and nematode faecal egg output in sheep. Vet. Zootec. 7, 127–133. Amarante, A.F.T., Bricarello, P.A., Rocha, R.A., Gennari, S.M., 2004. Resistance of Santa Ines, Suffolk and Ile de France lambs to naturally acquired gastrointestinal nematode infections. Vet. Parasitol. 120, 91–106. Amarante, A.F.T., Craig, T.M., Ramsey, W.S., Davis, S.K., Bazer, F.W., 1999a. Nematode burdens and cellular responses in the abomasal mucosa and blood of Florida Native, Rambouillet and crossbred lambs. Vet. Parasitol. 80, 311–324. Amarante, A.F.T., Craig, T.M., Ramsey, W.S., Sayed, N.M.E., Desouki, A.Y., Bazer, F.W., 1999b. Comparison of naturally acquired parasite burdens among Florida Native, Rambouillet and crossbred ewes. Vet. Parasitol. 85, 61–69. Bahirathan, M., Miller, J.E., Barras, S.R., Kearney, M.T., 1996. Susceptibility of Suffolk and Gulf Coast Native suckling lambs to naturally acquired strongylate nematode infection. Vet. Parasitol. 65, 259–268. Balic, A., Cunningham, C.P., Meeusen, E.N.T., 2006. Eosinophil interactions with Haemonchus contortus larvae in the ovine gastrointestinal tract. Parasite Immunol. 28, 107–115. Bricarello, P.A., Amarante, A.F.T., Rocha, R.A., Cabral Filho, S.L., Huntley, J.F., Houdijk, J.G.M., Abdalla, A.L., Gennari, S.M., 2005. Influence of dietary protein supply on resistance to experimental infections with Haemonchus contortus in Ile de France and Santa Ines lambs. Vet. Parasitol. 134, 99–109. Burke, J.M., Miller, J.E., 2002. Relative resistance of Dorper crossbred ewes to gastrointestinal nematode infection compared with St. Croix and Katahdin ewes in the southeastern United States. Vet. Parasitol. 109, 265–275. Costa, R.L.D., Bueno, M.S., Verı´ssimo, C.J., Cunha, E.A., Santos, L.E., Oliveira, S.M., Spo´sito Filha, E., Otsuk, I.P., 2007. Performance and nematode infection of ewe lambs on intensive rotational grazing with two different cultivars of Panicum maximum. Trop. Anim. Health Prod. 39, 255–263. Courtney, C.H., Parker, C.F., McClure, K.E., Herd, R.P., 1984. A comparison of the periparturient rise in fecal egg counts of exotic and domestic ewes. Int. J. Parasitol. 14, 377–381. Dawkins, H.J.S., Windon, R.G., Eagleson, G.K., 1989. Eosinophil responses in sheep selected for high and low responsiveness to Trichostrongylus colubriformis. Int. J. Parasitol. 19, 199–205. ESALQ, 2009. Posto Agrometeorolo´gico – A´rea de Fı´sica e Meteorologia – LCE – ESALQ – USP. http://www.esalq.usp.br/departamentos/lce/ postocon.html (accessed 02.02.09). Gamble, H.R., Zajac, A.M., 1992. Resistance of St. Croix lambs to Haemonchus contortus in experimentally and naturally acquired infections. Vet. Parasitol. 41, 211–225. Getachew, T., Dorchies, P., Jacquiet, P., 2007. Trends and challenges in the effective and sustainable control of Haemonchus contortus infection in sheep. Rev. Parasite 14, 3–14. Greer, A.W., 2008. Trade-offs and benefits: implications of promoting a strong immunity to gastrointestinal parasites in sheep. Parasite Immunol. 30, 123–132. Gruner, L., Aumont, G., Getachew, T., Brunel, J.C., Pery, C., Cognie, Y., Guerin, Y., 2003. Experimental infection of Black Belly and INRA 401 straight and crossbred sheep with trichostrongyle nematode parasites. Vet. Parasitol. 116, 239–249.
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Kanobana, K., Ploeger, H.W., Vervelde, L., 2002. Immune expulsion of the trichostrongylid Cooperia oncophora is associated with increased eosinophilia and mucosal IgA. Int. J. Parasitol. 32, 1389–1398. Keith, R.K., 1953. The differentiation of infective larvae of some common nematode parasites of cattle. Aust. J. Zool. 1, 223–235. Kemper, K.E., Elwin, R.L., Bishop, S.C., Goddard, M.E., Woolaston, R.R., 2009. Haemonchus contortus and Trichostrongylus colubriformis did not adapt to long-term exposure to sheep that were genetically resistant or susceptible to nematode infections. Int. J. Parasitol. 39, 607–614. Li, Y., Miller, J.E., Franke, D.E., 2001. Epidemiological observations and heterosis analysis of gastrointestinal nematode parasitism in Suffolk, Gulf Coast Native, and crossbred lambs. Vet. Parasitol. 98, 273–283. Mugambi, J.M., Audho, J.O., Njomo, S., Baker, R.L., 2005. Evaluation of the phenotypic performance of a Red Maasai and Dorper double backcross resource population: indoor trickle challenge with Haemonchus contortus. Vet. Parasitol. 127, 263–275. Mugambi, J.M., Bain, R.K., Wanyangu, S.W., Ihiga, M.A., Duncan, J.L., Murray, M., Stear, M.J., 1997. Resistance of four sheep breeds to natural and subsequent artificial Haemonchus contortus infection. Vet. Parasitol. 69, 265–273.
Ott, R.L., 1992. An Introduction to Statistical Methods and Data Analysis, 4 ed. Wadsworth Publishing Company, Belmont, p. 1051. Rocha, R.A., Amarante, A.F.T., Bricarello, P.A., 2004. Influence of reproduction status on susceptibility of Santa Ineˆs and Ile de France ewes to nematode parasitism. Small Rumin. Res. 55, 65–75. Rocha, R.A., Bresciani, K.D.S., Barros, T.F.M., Fernandes, L.H., Silva, M.B., Amarante, A.F.T., 2008. Sheep and cattle grazing alternately: nematode parasitism and pasture decontamination. Small Rumin. Res. 75, 135–143. Terefe, G., Grisez, C., Prevot, F., Bergeaud, J.P., Dorchies, P., Brunel, J.C., Franc¸ois, D., Fourquaux, I., Jacquiet, P., 2007. In vitro pre-exposure of Haemonchus contortus L3 to blood eosinophils reduces their establishment potential in sheep. Vet. Res. 38, 647–654. Ueno, H., Gonc¸alves, P.C., 1998. Manual para diagno´stico das helmintoses de ruminantes, 4 ed. Japan International Cooperation Agency, Tokyo, pp. 43. Van Vleck, L.D., Pollak, E.J., Oltenacu, E.A.B., 1987. Genetics for the Animal Sciences. W.H. Freeman, New York, p. 391. Walling, G.A., Wilson, A.D., Mcteir, B.L., Bishop, S.C., 2004. Increased heterozygosity and allele variants are seen in Texel compared to Suffolk sheep. Heredity 92, 102–109.
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Resistance of Santa Ines and crossbred ewes to naturally acquired gastrointestinal nematode infections A.F.T. Amarante a,*, I. Susin b, R.A. Rocha a, M.B. Silva a, C.Q. Mendes b, A.V. Pires b a b
UNESP - Universidade Estadual Paulista, Departamento de Parasitologia, Instituto de Biocieˆncias, Caixa Postal 510, Botucatu, SP, CEP 18618-000, Brazil USP – Universidade de Sa˜o Paulo, Departamento de Zootecnia – ESALQ, Piracicaba, SP, CEP 13418-900, Brazil
A R T I C L E I N F O
A B S T R A C T
Article history: Received 7 April 2009 Received in revised form 17 June 2009 Accepted 7 July 2009
This trial was carried out in Piracicaba, Sa˜o Paulo State, Brazil, to comparatively evaluate the degree of resistance to naturally acquired gastrointestinal nematode infections in sheep of the following genetic groups: purebred Santa Ines (SI), SI crossbred with Dorper (DO SI), Ile de France (IF SI), Suffolk (SU SI), and Texel (TE SI). Fifteen ewes from each group were raised indoors until 12 months of age. At this age, they were moved to pasture that was naturally contaminated by nematode infective larvae and were evaluated from December to May, 2007. Rainfall ranged from 267 mm in January to 37 mm in April. Maximum and minimum mean temperatures ranged from 32.5 8C to 19.0 8C in March and from 25.9 8C to 12.8 8C in May. There was an increase in the mean number of eggs per gram of feces (EPG) after animals were placed on pasture with significant difference between the SI (80 EPG) and IF SI (347 EPG) groups in January; and the DO SI (386 EPG) and TE SI (258 EPG) groups in May. The highest mean fecal egg count (FEC), 2073 EPG, was recorded for the TE SI group in February. All groups showed a progressive reduction in body weight throughout the experiment of 12.0% (TE SI) to 15.9% (SU SI). In general, the animals with the highest FEC presented the lowest packed cell volumes (PCV); the highest correlation coefficient between FEC PCV occurred in the SU SI sheep in January (r = 0.70; P < 0.01). Similarly, there was an inverse relationship between FEC and blood eosinophil values, with the highest correlation coefficient in the TE SI sheep in February (r = 0.64; P < 0.05). Immunoglobulin G (IgG) levels against Haemonchus contortus antigens increased in all groups as a result of the exposure to parasites and remained relatively constant until the end of the study, with the exceptions of SU SI and TE SI, which showed a rise in IgG levels during the last sampling that coincided with a reduction in mean FEC. In conclusion, crossbreeding Santa Ines sheep with any of the breeds evaluated can result in a production increase and the maintenance of a satisfactory degree of infection resistance, especially against H. contortus and Trichostrongylus colubriformis, the major nematodes detected in this flock. ß 2009 Elsevier B.V. All rights reserved.
Keywords: Haemonchus Trichostrongylus Genetic resistance Immune response Immunoglobulins IgG
1. Introduction Parasitic gastroenteritis is a major health problem for the sheep industry worldwide, causing great economical losses due to reduced productivity, mortality of animals,
* Corresponding author. Tel.: +55 14 3811 6239; fax: +55 14 3815 3744. E-mail address: [email protected] (A.F.T. Amarante). 0304-4017/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2009.07.009
and expenses with anthelmintics and labor. In addition, the frequent use of anthelmintics for the prophylaxis of gastrointestinal nematode (GIN) infections has led to the dissemination of populations of resistant parasites (Getachew et al., 2007). Alternative control measures include breeding strategies with the selection or the use of sheep breeds with resistance to GIN. This strategy has demonstrated sustainability, as strong evidence suggests that important sheep
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nematodes do not adapt to long-term exposure to sheep that are genetically resistant to GIN infections (Kemper et al., 2009). Some breeds of sheep demonstrate genetic resistance against Haemonchus contortus, the major parasite in tropical and sub-tropical areas; such breeds include the Florida Native (Amarante et al., 1999a) and Gulf Coast Native (Bahirathan et al., 1996; Li et al., 2001) sheep, raised in the south of the United States; St. Croix and Barbados Blackbelly (Courtney et al., 1984; Gamble and Zajac, 1992; Gruner et al., 2003), originating from the Caribbean islands; and the Red Maasai sheep from Africa (Mugambi et al., 1997). In Brazil, Santa Ines hair sheep demonstrate a greater resistance to GIN, when compared with some sheep breeds of European origin (Amarante et al., 2004; Rocha et al., 2004; Bricarello et al., 2005; Costa et al., 2007). These differences in breeds are usually the result of emphasis on different traits in selection, or of natural selection in breeds originating from different geographical locations. As such, problems with GIN infections in the sheep industry could be reduced by the use of resistant breeds; however, these resistant breeds can present inferior productivity when compared with other breeds selected for higher weight gain and meat quality. For example, the Ile de France is a breed notable for weight gain and carcass quality; however, its performance in tropical environments may not match that of tropical breeds adapted to the area, such as the Santa Ines hair sheep. Therefore, the farmer has to choose between a less productive breed that is well-adapted to the local environmental conditions and resistant to parasites or a highly productive breed that is not well-adapted to the climate and is more susceptible to parasites that occur in the tropics. A third option to solve such a problem is the crossbreeding of a susceptible breed, of elevated productivity, with a resistant, but less productive breed. The variation between breeds presents opportunities to combine and improve the characteristics of two different breeds in order to enhance productivity. In addition, crossbred progeny usually show heterosis in performance. Thus, crossbreeding allows the opportunity to obtain progress in a generation that would normally need several generations of selection to be reached (Van Vleck et al., 1987). At present, the Santa Ines sheep is predominant in most of the Brazilian territory due to its higher level of resistance to GIN (Amarante et al., 2004; Rocha et al., 2004; Bricarello et al., 2005; Costa et al., 2007). This trial was carried out to evaluate the degree of resistance of female sheep originating from the crossbreeding of Santa Ines ewes with Ile de France, Suffolk, Texel and Dorper sires, all breeds with a high potential for growth and meat production. 2. Materials and methods 2.1. Experimental area and animal management This study was carried out on an experimental farm located at the University of Sa˜o Paulo in Piracicaba, Brazil. The experimental area comprised 1 ha of grass pasture (Cynodon spp.) divided into a six-paddock rotational grazing system.
The average monthly relative humidity was always higher than 84% throughout the study. As usual for the region, rainfall was abundant in December, January and February (252 mm, 267 mm and 242 mm, respectively), with a total of 761 mm. In March (81 mm), April (37 mm) and May (58 mm), there was a decrease in precipitation. Maximum and minimum mean temperatures ranged from 32.5 8C to 19.0 8C in March 2007 and from 25.9 8C to 12.8 8C in May (ESALQ, 2009). Each experimental genotype group was composed of 15 young ewes, as follows: purebred Santa Ines (SI); crossbred Dorper Santa Ines (DO SI); crossbred Ile de France Santa Ines (IF SI); crossbred Suffolk Santa Ines (SU SI); and crossbred Texel Santa Ines (TE SI). The ewe lambs used in this trial were obtained by crossing 300 Santa Ines females with five sires of each of the following breeds: Santa Ines, Dorper, Texel, Suffolk and Ile de France. Each sire was exposed to the same number of females to assure genetic variability inside each experimental group. The birth date, the birth weight and the weight at weaning (60 days of age) were recorded, as well as whether the lambs were single or twins. This procedure allowed homogeneity for each genetic group. Animals were managed, according to routine procedures, at the sheep experimental farm. During late pregnancy, ewes were housed in pens with a concrete floor, where they were kept with their lambs until weaning. Ewes were vaccinated against clostridial disease (Sintoxan Polivalente1, Merial S.A., Uruguay), salmonelloses and pasteurelloses (Laborato´rio Prado S.A., Brazil) in the final third of the gestation. Lambs were vaccinated 15 days before weaning. Suckling lambs had free access to a concentrate with 18% crude protein in a creep feeding system. After weaning, lambs received a diet containing 16% crude protein, and consisting of hay (10%) and concentrate (90%). The concentrate was formulated from soybean meal, ground corn and mineral supplement. In order to prevent coccidiosis, monensin (25 ppm; Rumensin1, Elanco, Australia) was added to the diet. From 6 to 12 months of age, animals were fed on a higher roughage diet to avoid excessive fat deposition. Until the beginning of the trial, all animals were kept housed with free access to drinking water. At 12 months of age, the ewe lambs were released on pasture on December 19th, 2006, where they grazed, in a rotational system, as a single flock, in six paddocks until the end of the study. Pastures were expected to be naturally contaminated with infective larvae of GIN. In each paddock, ewe lambs had free access to water and mineral salt. The animals were weighed monthly and individual samples of blood and feces were taken. To avoid deaths, ewes were individually treated with levamisole (7.5 mg/ kg, Ripercol1, Fort Dodge, Brazil) whenever their fecal egg counts (FEC) were higher than 4000 eggs per gram (EPG) or their packed cell volume (PCV) was lower than 20% (Amarante et al., 1999b). During the last two months of the study, two tracer lambs, free of infections of GIN, were placed together with the experimental animals. At the end of the study, these tracer lambs were sacrificed in order to identify species of helminths present in the flock.
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2.2. Parasitology Fecal samples were taken directly from the rectum of the animals for fecal egg count (FEC) determination using a modified McMaster technique (Ueno and Gonc¸alves, 1998). Each egg counted represented 100 eggs/g. Composite fecal cultures were performed separately for each group of sheep for production of third stage larvae, which were identified according to the descriptions of Keith (1953). At necropsy of the two tracer lambs, the abomasum and the small and large intestines were removed, opened and the contents placed in graduated buckets. A 10% aliquot was preserved in 4% formalin for subsequent enumeration of the helminths and identification to species level of the adult male nematodes, based on the morphology of their spicules (Ueno and Gonc¸alves, 1998). Pasture samples were also collected on the day of fecal and blood collection to assess the number of infective GIN larvae per kilogram of dry matter (L3/kg DM) from two of the paddocks grazed by the experimental animals. For pasture sampling, the grass collector followed a W-shaped track in each paddock, collecting samples every four steps (approximately 3.5 m). Grass was cut as close to the ground as possible and the samples were processed in the laboratory, as described by Amarante and Barbosa (1995).
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protease inhibitor solution (Complete Mini Solution1, Roche, USA) using an Ultra Turrax1 (Ika, Germany). The extract was centrifuged (15,000 g) at 4 8C for 20 min and the supernatant containing the adult-soluble-antigen extract was collected. Protein concentrations of L3 and adult antigens were determined using a kit (Protal me´todo colorime´trico1, Laborlab, Brazil) and absorbance was read at 562 nm. The antigen extracts were stored in aliquots at 80 8C until further use. 2.6. Parasite-specific serum IgG
Blood samples with EDTA and without anti-coagulant were collected by jugular veni-puncture. Packed cell volume (PCV) was determined by the micro-hematocrit method. Serum samples were stored at 20 8C until use for immunoglobulin measurements. The eosinophil counts in peripheral blood were made in a Neubauer’s chamber after staining with Carpentier’s solution (Dawkins et al., 1989); counts were expressed as the number of cells per ml of blood.
Polystyrene micro-titer plates (Nunc, USA) were coated with 100 ml of adult or L3 H. contortus (2 mg/ml) antigens diluted in carbonate–bicarbonate buffer (pH 9.6); plates were incubated overnight at 25 8C. All subsequent incubations were carried out for 1 h at 37 8C using, in each well, a total of 100 ml of reagents. Between each step, plates were washed three times with ultra pure water (EASYpure II UV, Barnstead, USA) containing 0.05% Tween 20. After coating, blocking was carried out with 0.1% Gelatin (Amresco, USA) and 0.05% Tween 20 (Amresco, USA) in PBS (PBS–GT). Serum samples were diluted in PBS– GT (1:1000) and applied in duplicate. Plates were then incubated with rabbit polyclonal antibody to sheep IgG (Abcam Inc. UK; 1:2000 in PBS–GT) followed by polyclonal goat anti-rabbit immunoglobulins linked to alkaline phosphatase (Dako, Denmark; 1:2000 in PBS–GT). Finally, substrate solution (pnitrophenyl phosphate disodium salt, PNPP, Pierce, USA) was added to each well and the enzymatic reaction was allowed to proceed at room temperature for 30 min and plates were immediately read using an automated ELISA reader (Biotrak II, AmershamBiosciences, UK) at 405 nm. The standard positive serum was obtained from a sheep repeatedly infected with H. contortus. Results were expressed as the percentage of the optical density value of the positive-reference serum (Kanobana et al., 2002).
2.4. Enzyme-linked immunosorbent assay
2.7. Statistical analysis
Immunoglobulin G (IgG) levels against larval and adult H. contortus antigens were measured in serum samples.
Significant differences between groups for FEC, PCV, body weight, eosinophils and IgG levels were assessed by oneway analysis of variance using the statistical software, Minitab 11.21 (Minitab Inc., USA). Group means were compared using the least significant difference at P < 0.05 (Ott, 1992). FEC data were transformed using log10(x + 1) prior to analysis. The figures in the results are expressed as arithmetic means (standard error of the mean). Spearman’s correlation coefficient between variables was assessed using the same software.
2.3. Hematology
2.5. Larval and adult H. contortus antigens Infective H. contortus third stage larvae (L3) were obtained from fecal cultures of monospecifically infected donor sheep. The same donor sheep was sacrificed and adult worms were recovered from the abomasum and washed several times in PBS. H. contortus L3 were exsheathed, under microscopic monitoring, with 0.1% sodium hypochlorite solution and subsequently washed in PBS. Larvae were fragmented at 4 8C in PBS supplemented with protease inhibitor solution (Complete Mini Solution, Roche, USA) using an ultrasonic processor (VibraCell–Sonics & Materials Inc., USA). The homogenized content was centrifuged at 15,000 g, at 4 8C, for 20 min and the supernatant containing the L3-solubleantigen extract was collected. Adult H. contortus worms were fragmented at 4 8C in PBS supplemented with 2 ml of
3. Results At the beginning of the trial, some sheep were passing nematode eggs in feces, but in a small number. The maximum FEC was 900 EPG in one SU SI ewe (Table 1). These findings demonstrated that, while housed, young ewes were exposed to an apparently low level of infective larval (L3) contamination. After being introduced to their grazing paddocks, the ewes started to present a higher
TE SI (n = 15)
93.3 (0; 500), 0.79ab 0.30 1257.0 (0; 12,600), 1.64ab 0.41 2073.0 (0; 14,700), 1.83a 0.43 260.0 (0; 1220), 1.34a 0.36 980.0 (0; 4400), 2.19a 0.32 258. 0 (0; 1200), 1.15b 0.41
SU SI (n = 15)
246.7 (0; 900), 1.90a 0.26 1007.0 (0; 4500), 1.62ab 0.41 720.0 (0; 2900), 2.21a 0.31 253.3 (0; 1300), 1.72a 0.29 1485.0 (0; 14,200), 1.57a 0.44 342.0 (0; 1100), 1.87ab 0.34 6.7 (0; 100), 0.13b 0.13 347.0 (0; 2200), 1.95b 0.28 1227.0 (0; 7500), 2.08a 0.36 146.7 (0; 400), 1.54a 0.29 1086.0 (0; 6000), 2.17a 0.35 527.0 (0; 4500), 1.64ab 0.34 6.7 (0; 100), 0.13b 0.13 146.7 (0; 600), 1.26ab 0.32 1453.0 (0; 9500), 2.26a 0.33 333.0 (0; 1500), 1.93a 0.28 940.0 (0; 5500), 1.84a 0.37 386.0 (0; 1600), 2.19a 0.26
IF SI (n = 15) DO SI (n = 15) SI (n = 15)
146.7 (0; 600), 1.14a 0.26 80.0 (0; 400), 0.77a 0.29 320.0 (0; 1800), 1.65a 0.33 266.7 (0; 1500), 1.89a 0.27 240.0 (0; 1500), 1.70a 0.29 814.0 (0; 9300), 1.78ab 0.34
Data
19/December 16/January 13/February 15/March 11/April 10/May
Table 1 Fecal egg counts of the Santa Ines (SI), Dorper Santa Ines (DO SI), Ile de France Santa Ines (IF SI), Suffolk Santa Ines (SU SI) and Texel Santa Ines (TE SI) young ewes during natural infection by gastrointestinal nematodes.
Results are arithmetic mean with minimum and maximum values in parenthesis. Means of log transformed values (standard error) followed by different superscripts in the same line are significantly different (P < 0.05).
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Fig. 1. Means of packed cell volume (A) and blood eosinophils (B) of the Santa Ines (SI) purebred sheep and Dorper Santa Ines (DO SI), Ile de France Santa Ines (IF SI), Suffolk Santa Ines (SU SI) and Texel Santa Ines (TE SI) crossbred sheep during natural infection by gastrointestinal nematodes. Bars represent standard error of the mean.
degree of infection. Significant difference among group means occurred between SI (80 EPG) and IF SI (347 EPG) groups in January, and between DO SI (386 EPG) and TE SI (258 EPG) in May. The highest FEC mean value (2073 EPG) was recorded in TE SI sheep in February (Table 1). The PCV averages showed a progressive reduction throughout the experiment (Fig. 1). Significant differences between group means were registered only at the beginning of the study: the average of the animals SI (33.6 0.52%) was lower than the average of the DO SI (36.5 1.04%) and IF SI (36.3 0.59%) in December, and the averages of the SU SI (29.9 1.52%) and TE SI (30.2 1.43%) groups were significantly lower than the averages of the SI (33.6 0.65%), DO SI (34.6 1.06%) and IF SI (34.3 0.61%) groups in January. In order to avoid cases of mortality, the animals were treated individually with anthelmintics when they presented FEC higher than 4000 EPG or PCV values lower than 20%. There was no case of any animal needing more than one treatment, with the exception of one SU SI ewe. The animals treated were as follows: one SI ewe (in May), two DO SI (both in February), three IF SI (February, April and May), two SU SI (one in January and other twice, in January and in April) and five TE SI (one in January, three in February and one in April). The percentage reduction in FEC was greater than 93% at seven days after the administration of levamisole. The lowest eosinophil averages occurred at the beginning of the study. In the following months, average counts increased in all groups and were usually higher than 1000 cells/ml (Fig. 1). During the last month of the study, the averages of the SI and TE SI groups were significantly lower than those of the other groups.
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Table 2 Mean levels expressed as a percent of standard serum (standard error) of IgG against antigens of third stage larvae (L3) and adults of Haemonchus contortus in the serum of the Santa Ines (SI), Dorper Santa Ines (DO SI), Ile de France Santa Ines (IF SI), Suffolk Santa Ines (SU SI) and Texel Santa Ines (TE SI) young ewes during natural infections by gastrointestinal nematodes. Date
Antigen
DO SI (n = 15)
IF SI (n = 15)
SU SI (n = 15)
TE SI (n = 15)
19/December
L3 Adult
SI (n = 15) 54.7a 3.86 33.5a 2.57
64.2a 5.74 48.3a 6.37
49.0a 3.88 28.7b 3.65
92.3b 6.15 87.2c 5.54
82.5b 6.79 79.0c 5.77
16/January
L3 Adult
112.0a 3.75 101.9a 4.33
101.3ab 5.3 100.9a 5.46
85.6b 5.76 88.5a 6.05
99.0ab 7.35 110.3a 7.51
91.2b 6.39 93.8a 6.94
13/February
L3 Adult
104.9a 4.46 97.2ac 3.92
99.6a 6.14 87.8a 5.45
94.2a 6.09 86.1a 5.22
102.4a 5.45 112.8b 3.80
96.6a 5.92 103.3bc 4.16
15/March
L3 Adult
111.9ac 2.58 111.4a 3.01
109.1ac 4.05 103.7a 4.74
94.8 b 3.76 94.8a 6.15
115.2c 3.55 108.6a 3.97
102.8ab 5.64 98.3a 4.43
11/April
L3 Adult
119.2a 3.94 117.8a 4.24
116.6a 3.74 120.9a 2.99
101.7b 3.42 104.9b 6.63
117.3a 4.45 119.6a 4.88
100.2b 4.77 104.1b 3.57
10/May
L3 Adult
105.6a 1.55 104.1a 2.59
104.1ab 2.43 102.7a 2.91
98.2b 1.87 103.8a 2.16
149.4c 3.13 147.6b 3.89
138.6d 2.39 133.9c 3.70
Means followed by different superscripts in the same line are significantly different (P < 0.05).
Analysis of immunoglobulin concentrations in the serum indicated that IgG means against H. contortus antigens were considerably higher in the SU SI and in the TE SI groups at the beginning and at the end of the study (Table 2). The lowest means were detected in the first sampling, reflecting the low exposure of the animals to GIN, while they were housed. With regard to the groups, the lowest means of IgG against L3 were recorded in the IF SI, except on April 11th. The same occurred for the adult antigens: IF SI presented the lowest means, except for the last sampling (May 10). Correlation coefficients between IgG against L3 and IgG against adult antigens were high in all samplings (r from 0.764 to 0.922; P < 0.001). With regard to body weight, the SI animals were the lightest, while the DO SI and TE SI groups presented intermediate body weight averages and the SU SI and IF SI groups were the heaviest (Table 3). There was a progressive reduction in average weight until the end of the experiment. At the end of the experiment, the animals had lost, on average, from 12.5% (TE SI) to 15.9% (SU SI) of their body weight, compared with their initial weight. These two group mean values were significantly different. Haemonchus spp. infective larvae were predominant in fecal cultures with percentages ranging from 42% to 94% in the DO SI group; 64% to 92% in IF SI; 45% to 92% in SI; 34% to 91% in SU SI; and 40% to 92% in TE SI. The
highest Trichostrongylus spp. percentages (51%) occurred in the SI Group on March 15th. Cooperia spp. and Oesophagostomum spp. infective larvae were detected, although in low percentages; the highest values were, respectively, observed in cultures from the DO SI group in March (15%) and in May (24%). Eggs of Strongyloides spp. were frequently detected in fecal samples collected from February to May, but in low numbers. The highest mean Strongyloides spp. FEC was recorded in DO SI ewes in May (235.7 EPG). Besides nematode eggs, oocysts of Eimeria spp. and eggs of Moniezia spp. were also found in some of the fecal examinations. Haemonchus spp. and Trichostrongylus spp. third stage larvae were found in pasture (Fig. 2). The peak in grass contamination occurred in April, when 703.5 Haemonchus spp. L3/kg DM and 309.1 Trichostrongylus spp. L3/kg DM were recorded. Three nematode species were recovered from tracer animals, with the following mean number of worms: 1200 H. contortus, 3121 Trichostrongylus colubriformis and 240 Cooperia curticei. In general, the animals from each group with the highest FEC presented the lowest PCV values and blood eosinophils, corresponding to negative correlation coefficients (Table 4). The highest correlation coefficients between FEC IgG against adult H. contortus antigens
Table 3 Mean (standard error) body weight (kg) of the Santa Ines (SI), Dorper Santa Ines (DO SI), Ile de France Santa Ines (IF SI), Suffolk Santa Ines (SU SI) and Texel Santa Ines (TE SI) young ewes during natural infection by gastrointestinal nematodes. Data
SI (n = 15)
DO SI (n = 15)
IF SI (n = 15)
SU SI (n = 15)
TE SI (n = 15)
19/December 16/January 13/February 15/March 11/April 10/May Weight reduction (kg)a Weight reduction (%)a
50.1a 1.18 45.2a 1.21 45.5a 1.27 47.1a 1.24 46.1a 1.31 43.6a 1.36 6.5a 0.65 13.2ab 1.38%
56.5b 1.26 52.3b 1.13 52.0b 1.11 51.8b 1.15 51.2b 1.24 48.4b 1.35 8.1a 0.80 14.4ab 1.36%
61.6c 1.58 57.5c 1.64 57.3c 1.50 56.0cd 1.51 56.7c 1.45 53.8c 1.26 7.8a 0.65 12.5ab 0.90%
65.4c 1.98 59.1c 1.78 57.7c 1.61 57.4 d 1.47 57.3c 1.51 54.8c 1.36 10.6b 1.03 15.9a 1.21%
56.5b 1.39 52.0b 1.26 51.8b 1.37 53.1bc 1.07 52.4b 1.22 49.6b 1.07 6.9a 0.75 12.0b 1.20%
Means followed by different superscripts in the same line are significantly different (P < 0.05). a Weight reduction = last body weight initial body weight.
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4. Discussion
Fig. 2. Mean Haemonchus spp. and Trichostrongylus spp. third stage larvae by kilogram of herbage dry matter (L3/kg DM) recovered from pastures grazed by the experimental sheep.
were recorded in TE SI group in the last two months of the trial (respectively, r = 0.77 and 0.78; P < 0.01). Correlation coefficients between FEC IgG against L3 antigens were usually lower, the maximum value (r = 0.57; P < 0.05) was found in the SI group in April. All correlation coefficients between FEC and IgG were negative and several were statistically significant in April (Table 4). Correlation coefficients between FEC and body weight were low (Table 4).
Table 4 Correlation coefficients between fecal egg count (FEC) and the following variables: packed cell volume (PCV), number of eosinophils in blood (Eos), body weight (BW), IgG against L3 (IgG-L3) and adult (IgG-adult) H. contortus antigens of the Santa Ines (SI), Dorper Santa Ines (DO SI), Ile de France Santa Ines (IF SI) and Suffolk Santa Ines (SU SI), naturally infected with gastrointestinal nematodes. Date
Group
16/January
SI SI DO SI IF SI SU SI TE
0.31 0.49 0.35 0.70** 0.66*
0.31 0.11 0.43 0.11 0.09
13/February
SI SI DO SI IF SI SU SI TE
0.17 0.58* 0.50 0.38 0.65**
15/March
SI SI DO SI IF SI SU SI TE
11/April
10/May
* **
P < 0.05. P < 0.01.
FEC PCV
FEC BW
FEC Eos
FEC IgG-L3
FEC IgG-Ad
0.58* 0.08 0.42 0.62* 0.39
0.04 0.44 0.41 0.04 0.54*
0.13 0.35 0.45 0.13 0.66**
0.13 0.08 0.06 0.56* 0.18
0.51 0.16 0.16 0.09 0.64*
0.22 0.11 0.55* 0.12 0.05
0.23 0.35 0.44 0.08 0.02
0.45 0.26 0.25 0.31 0.26
0.26 0.20 0.41 0.10 0.41
0.45 0.07 0.55 0.10 0.24
0.12 0.30 0.17 0.27 0.27
0.35 0.01 0.09 0.07 0.63*
SI SI DO SI IF SI SU SI TE
0.01 0.38 0.51 0.32 0.25
0.22 0.02 0.25 0.21 0.28
0.21 0.63* 0.12 0.39 0.50
0.57* 0.56* 0.26 0.40 0.52*
0.36 0.25 0.28 0.72** 0.77**
SI SI DO SI IF SI SU SI TE
0.42 0.02 0.16 0.14 0.53
0.08 0.38 0.10 0.48 0.03
0.33 0.41 0.16 0.57 0.49
0.15 0.13 0.32 0.33 0.33
0.08 0.20 0.07 0.21 0.78**
All animals, raised indoors since birth, receiving a balanced diet and were exposed to a low level of GIN infection, were in excellent conditions at the beginning of the study. After the first sampling, the animals were kept exclusively on pasture, and were exposed to a higher level of parasitic infection, as well to the environmental conditions, characterized in the region, by a summer with high rainfall and temperature. As a consequence of the infections, the change in food supply and, possibly, as a result of exposure to the weather conditions, the animals lost weight, associated with a decrease in PCV and increase in FEC. The changes in these variables were possibly caused, in part, by GIN infections, especially H. contortus, a blood sucking parasite and the major species found in the flock, followed by T. colubriformis. These are the major sheep nematodes in the region (Amarante et al., 2004; Rocha et al., 2008). An increase in numbers of infective larvae on pasture occurred in the autumn, associated with a reduction in rainfalls. These findings are also in agreement with another study carried out in the region (Amarante et al., 2004) and suggest that the heavy summer rains can remove larvae from the herbage. The acquisition and maintenance of immunity to gastrointestinal parasites in sheep is a nutritionally costly process that induces a relatively short-term diversion of nutrients from productive to immunological tissues (reviewed by Greer, 2008). The costs for the development of immune protection against the GIN parasites might have been another reason for the reduction in body weight of the animals in the present study. The resistance was not uniform among animals, even in the same group. Of the 15 TE SI ewes, five needed one anthelmintic treatment due to the presentation of high FEC and/or low PCV, while the other animals of the same group (10 ewes) remained healthy. In the other groups, a smaller number of animals needed anthelmintic treatment: three IF SI, two SU SI, two DO SI and only one SI. After being treated, animals recovered and developed a degree of resistance that was apparently effective for avoiding the establishment of a new heavy re-infection. The exception was one SU SI ewe that was treated twice, in January and in April. Several studies have shown that the Santa Ines hair sheep are more resistant to GIN infections than wool sheep such as Suffolk and Ile de France breeds (Amarante et al., 2004; Rocha et al., 2004; Bricarello et al., 2005; Costa et al., 2007). In the present study, with few exceptions, the F1 animals produced by crossing both susceptible breeds (Suffolk or Ile de France) with Santa Ines animals, resulted in hybrids with a degree of resistance to GIN infections similar to that of the parental resistant breed. The same occurred with SI DO crossbred animals, and to a lesser extent in the SI TE group. The results of other studies carried out with animals produced by the crossing of resistant with susceptible breeds have demonstrated that the degree of resistance of the hybrids can vary as a function of the breeds evaluated, the age of the animals and whether the evaluations were from natural or artificial infections. As such, different
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results were reported in sheep produced by crossing Florida Native (resistant) and Rambouillet (susceptible) breeds. In the evaluations with adult ewes, the F1s showed an elevated degree of resistance to natural infections, similar to that shown by the resistant parental Florida Native breed (Amarante et al., 1999a). Conversely, in lambs artificially infected with H. contortus, the F1s presented a worm burden that was similar to the susceptible Rambouillet lambs (Amarante et al., 1999b). Similarly, in an evaluation of the phenotypic performance of a Red Maasai and Dorper double backcross resource population, Mugambi et al. (2005) observed that the Red Maasai do not respond to artificial challenge with H. contortus in the way that they do to natural infection, suggesting that phenotyping of Red Maasai and its cross-lambs, for purposes of selecting those that are resistant or susceptible, is best done under natural challenge. As observed in the present study, crossbred animals of the Suffolk and Dorper breeds also showed a relatively high degree of resistance in studies carried out in the USA. Heterosis analysis showed that FEC, PCV, nematode count and weight gain of the crossbred Suffolk and Gulf Coast Native lambs favored the Native group (Li et al., 2001); and the relative resistance of mature Dorper crossbred (Dorper Romanov or Dorper Romanov St. Croix) ewes was comparable to that of Katahdin and St. Croix ewes and superior to that of Hampshire ewes under natural or artificial infection (Burke and Miller, 2002). As a consequence of exposure to nematode larvae on pasture, an increase in IgG levels and eosinophilia was evident in all groups. The development of eosinophilia is a characteristic feature of helminth infection and there is strong evidence of an important role of eosinophils in the defense against infective larvae of GIN (Balic et al., 2006; Terefe et al., 2007). One month of exposure to parasites on pasture resulted in an elevation of IgG levels, which remained relatively constant until the end of the study, with the exception of the SU SI and TE SI groups, which showed a rise in IgG in the last sampling, which coincided with a reduction in mean FEC. Analysis of the coefficients of correlation demonstrated that the levels of antibodies had a greater influence on FEC of the TE SI animals, in comparison with other groups. During the last three months of the study, the TE SI animals showed significant negative correlation coefficients between FEC and the levels of IgG against adult antigens of H. contortus, i.e., animals that produced more antibodies were more resistant to GIN parasites. The TE SI group also had the highest number of animals that needed individual anthelminthic treatment (5 of 15 ewes). These results indicate that the animals with the Texel background had a higher genetic variability, which is in agreement with the description by Walling et al. (2004), who found increased heterozygosity and allele variants in Texel, compared to Suffolk sheep. Comparatively, crossbred young ewes presented a similar resistance to GIN infections to that of their purebred Santa Ines counterparts and were heavier. Therefore, all breeds tested can be used with good results in crossing programs with Santa Ines sheep. Other studies
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are necessary to evaluate the performance of crossbred ewes used in a ‘‘three-cross’’ system. In conclusion, crossbreeding of Santa Ines sheep with breeds with a high potential of production provides promise as a measure to increase sheep production, with the maintenance of a satisfactory degree of resistance to gastrointestinal nematode parasite infection. Acknowledgements This study was funded by Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FAPESP). A.F.T. Amarante, I. Susin and A.V. Pires received support from CNPq and R.A. Rocha, C.Q. Mendes and M.B. Silva from FAPESP. References Amarante, A.F.T., Barbosa, M.A., 1995. Seasonal variations in populations of infective larvae on pasture and nematode faecal egg output in sheep. Vet. Zootec. 7, 127–133. Amarante, A.F.T., Bricarello, P.A., Rocha, R.A., Gennari, S.M., 2004. Resistance of Santa Ines, Suffolk and Ile de France lambs to naturally acquired gastrointestinal nematode infections. Vet. Parasitol. 120, 91–106. Amarante, A.F.T., Craig, T.M., Ramsey, W.S., Davis, S.K., Bazer, F.W., 1999a. Nematode burdens and cellular responses in the abomasal mucosa and blood of Florida Native, Rambouillet and crossbred lambs. Vet. Parasitol. 80, 311–324. Amarante, A.F.T., Craig, T.M., Ramsey, W.S., Sayed, N.M.E., Desouki, A.Y., Bazer, F.W., 1999b. Comparison of naturally acquired parasite burdens among Florida Native, Rambouillet and crossbred ewes. Vet. Parasitol. 85, 61–69. Bahirathan, M., Miller, J.E., Barras, S.R., Kearney, M.T., 1996. Susceptibility of Suffolk and Gulf Coast Native suckling lambs to naturally acquired strongylate nematode infection. Vet. Parasitol. 65, 259–268. Balic, A., Cunningham, C.P., Meeusen, E.N.T., 2006. Eosinophil interactions with Haemonchus contortus larvae in the ovine gastrointestinal tract. Parasite Immunol. 28, 107–115. Bricarello, P.A., Amarante, A.F.T., Rocha, R.A., Cabral Filho, S.L., Huntley, J.F., Houdijk, J.G.M., Abdalla, A.L., Gennari, S.M., 2005. Influence of dietary protein supply on resistance to experimental infections with Haemonchus contortus in Ile de France and Santa Ines lambs. Vet. Parasitol. 134, 99–109. Burke, J.M., Miller, J.E., 2002. Relative resistance of Dorper crossbred ewes to gastrointestinal nematode infection compared with St. Croix and Katahdin ewes in the southeastern United States. Vet. Parasitol. 109, 265–275. Costa, R.L.D., Bueno, M.S., Verı´ssimo, C.J., Cunha, E.A., Santos, L.E., Oliveira, S.M., Spo´sito Filha, E., Otsuk, I.P., 2007. Performance and nematode infection of ewe lambs on intensive rotational grazing with two different cultivars of Panicum maximum. Trop. Anim. Health Prod. 39, 255–263. Courtney, C.H., Parker, C.F., McClure, K.E., Herd, R.P., 1984. A comparison of the periparturient rise in fecal egg counts of exotic and domestic ewes. Int. J. Parasitol. 14, 377–381. Dawkins, H.J.S., Windon, R.G., Eagleson, G.K., 1989. Eosinophil responses in sheep selected for high and low responsiveness to Trichostrongylus colubriformis. Int. J. Parasitol. 19, 199–205. ESALQ, 2009. Posto Agrometeorolo´gico – A´rea de Fı´sica e Meteorologia – LCE – ESALQ – USP. http://www.esalq.usp.br/departamentos/lce/ postocon.html (accessed 02.02.09). Gamble, H.R., Zajac, A.M., 1992. Resistance of St. Croix lambs to Haemonchus contortus in experimentally and naturally acquired infections. Vet. Parasitol. 41, 211–225. Getachew, T., Dorchies, P., Jacquiet, P., 2007. Trends and challenges in the effective and sustainable control of Haemonchus contortus infection in sheep. Rev. Parasite 14, 3–14. Greer, A.W., 2008. Trade-offs and benefits: implications of promoting a strong immunity to gastrointestinal parasites in sheep. Parasite Immunol. 30, 123–132. Gruner, L., Aumont, G., Getachew, T., Brunel, J.C., Pery, C., Cognie, Y., Guerin, Y., 2003. Experimental infection of Black Belly and INRA 401 straight and crossbred sheep with trichostrongyle nematode parasites. Vet. Parasitol. 116, 239–249.
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