Effect of growing forage legumes on the migration and survival in the pasture of gastrointestinal nematodes of sheep.

In order to identify types of forage that inhibit pasture contamination, an evaluation was performed of the effect of the forage legumes Trifolium repens (white clover), Trifolium pratense (red clover) and Lotus corniculatus (bird's-foot-trefoil) on the survival and migration of infective larvae (L3) of gastrointestinal nematodes (GIN) of sheep. An experimental area of 441 m2 was divided into four blocks, subdivided into areas of 1.20 × 1.20 in which the three forage legumes were separately overseeded. After growth of the forage in each subdivision, experimental units were established that were later artificially contaminated with sheep faeces containing GIN eggs. Between October and December 2018, pasture, faecal and soil samples were collected on four occasions during weeks 1, 2, 4 and 8 after the deposition of faeces. In week 6, the forage legumes in all the experimental units were mown to simulate grazing. The number of L3 was quantified to determine their survival in the pasture, faeces and soil. In addition, the horizontal migration of L3 was measured at two distances from the faecal pellets (10 and 30 cm), as well as their vertical migration at two heights of the plant stems, that is, lower half and upper half. Larvae vertical migration was affected by the forage species (P < 0.001), in that bird's-foot-trefoil contained fewer larvae in the upper stratum. Bird's-foot-trefoil restricted the migration of L3 to the upper stratum of the plant, which could potentially decrease the risk of infection by intestinal nematodes in grazing sheep.

Influence of protein supplementation during late pregnancy and lactation on the resistance of Santa Ines and Ile de France ewes to Haemonchus contortus.

This experiment aimed to evaluate the effect of periparturient metabolizable protein (MP) nutrition on resistance to Haemonchus contortus in single rearing Ile de France and Santa Ines ewes. The restriction-fed iso-energetic diet was calculated to provide either 0.8 (low MP diet) or 1.3 (high MP diet) times MP, from three weeks before parturition until eight weeks into lactation. The ewes were experimentally infected with 1000 H. contortus infective larvae (L3) three times a week (Mondays, Wednesdays and Fridays), starting five weeks before the predicted date for parturition until a total of 15,000 L3 had been administered. While both breeds showed elevated fecal egg counts (FEC), these values were significantly lower for Santa Ines ewes than Ile de France ewes, but were independent of level of MP feeding. The latter also did not affect lamb weight gain and ewe body weight variation in each breed. Packed cell volume and total plasma protein for Santa Ines in all periods were significantly higher than those for Ile de France ewes (P<0.01) but were not affected by nutrition. In contrast, levels of serum IgG and IgA antibodies against somatic H. contortus infective larvae and adult antigens were similar between breeds but higher in animals that received high MP diets (P<0.05). The reduced body score of ewes at the beginning of the experiment probably influenced their high susceptibility to incoming larvae. Since, unexpectedly, MP scarcity was not achieved in this experiment, our data support the view that Santa Ines ewes are more resistant to H. contortus than Ile de France ewes.

Cytokine gene expression in response to Haemonchus placei infections in Nelore cattle.

This study aimed to evaluate the expression of a subset of cytokine genes in response to Haemonchus placei infections in Nelore cattle presenting different degrees of resistance to natural infections. One hundred weaned bulls, initially 11-12 months old, were evaluated and kept on the same pasture. Faecal and blood samples were collected for parasitological and immunological assays. The seven most resistant and the eight most susceptible animals were selected based on nematode faecal egg counts (FEC) and worm burden. Serum was collected to measure antibody titres, and abomasum and abomasal lymph node tissue samples were collected to analyse the expression of a subset of cytokine genes (IL-2, IL-4, IL-8, IL-12p35, IL-13, TNF-alpha, IFN-gamma, MCP-1, MCP-2, MUC-1) using real-time RT-PCR. Mast cells, eosinophils and globule leukocytes in the abomasal mucosa were enumerated, and IgA levels in the mucus were assessed. Gene expression analysis in the abomasal tissue indicated that IL-4 and IL-13 (TH2 cytokines) were up-regulated in the resistant group, whereas TNF-alpha (TH1/TH2 cytokine) was up-regulated in the susceptible group. In abomasal lymph nodes, IL-4 and IFN-gamma were up-regulated in the resistant and susceptible groups, respectively. In the resistant group, serum IgG1 levels were higher against antigens of H. placei infective larvae on days 14, 42, 70 and 84 and against antigens of H. placei adults on day 84 (P<0.05). The resistant group had higher mast cell counts in the abomasal mucosa than the susceptible group (P<0.05). These results indicate a protective TH2-mediated immune response against H. placei in the resistant group and a less protective TH1 response in the susceptible group.

Immunological responses and cytokine gene expression analysis to Cooperia punctata infections in resistant and susceptible Nelore cattle.

Cellular and humoral immune response, as well as cytokine gene expression, was assessed in Nelore cattle with different degrees of resistance to Cooperia punctata natural infection. One hundred cattle (male, weaned, 11-12 months old), kept together on pasture, were evaluated. Faecal and blood samples were collected for parasitological and immunological assays. Based on nematode faecal egg counts (FEC) and worm burden, the seven most resistant and the eight most susceptible animals were selected. Tissue samples of the small intestine were collected for histological quantification of inflammatory cells and analysis of cytokine gene expression (IL-2, IL-4, IL-8, IL-12p35, IL-13, TNF-alpha, IFN-gamma, MCP-1, MCP-2, and MUC-1) using real-time RT-PCR. Mucus samples were also collected for IgA levels determination. Serum IgG1 mean levels against C. punctata antigens were higher in the resistant group, but significant differences between groups were only observed 14 days after the beginning of the experiment against infective larvae (L3) and 14 and 84 days against adult antigens. The resistant group also presented higher IgA levels against C. punctata (L3 and adult) antigens with significant difference 14 days after the beginning of the trial (P<0.05). In the small-intestine mucosa, levels of IgA anti-L3 and anti-adult C. punctata were higher in the resistant group, compared with the susceptible group (P<0.05). Gene expression of both T(H)2 cytokines (IL-4 and IL-13) in the resistant group and T(H)1 cytokines (IL-2, IL-12p35, IFN-gamma and MCP-1) in the susceptible group was up-regulated. Such results suggested that immune response to C. punctata was probably mediated by T(H)2 cytokines in the resistant group and by T(H)1 cytokines in the susceptible group.

Field study on nematode resistance in Nelore-breed cattle.

The present study evaluated Nelore cattle with different degrees of resistance to natural infections by gastrointestinal nematodes. One hundred weaned male cattle, 11-12 months of age, were kept on the same pasture and evaluated from October 2003 to February 2004. Faecal and blood samples were collected for parasitological, haematological and immunological tests. In February 2004, the 10 most resistant and the 10 most susceptible animals were selected based on individual means of nematode faecal egg counts (FEC). Such animals were slaughtered for worm burden determination and nematode species identification. The repeatability estimates for FEC (+/-S.D.), log-transformed FEC and packed-cell volume (PCV) in all animals were 0.3 (+/-0.05), 0.26 (+/-0.04) and 0.42 (+/-0.05), respectively. The resistant group showed lower FEC and worm burdens than the susceptible group (P<0.05). There were no significant differences between groups regarding mean body weight, weight gain, PCV and total serum protein values (P>0.05). The resistant group showed higher total serum IgE levels (P<0.05) and higher mean eosinophil blood counts. However, the latter was statistically significant only 42 days after the beginning of the study. Nematodes Cooperia punctata and Haemonchus placei were predominant and the correlation between Cooperia and Haemonchus burdens was 0.64 (P<0.05), which indicated that animals presenting increased numbers of one of those genera probably had increased numbers of the other. The current study provides further evidence of IgE active role in nematode immunity and suggests that total serum IgE level might serve as an additional marker to select Nelore cattle that are responsive to H. placei and C. punctata infections.

Influence of dietary protein supply on resistance to experimental infections with Haemonchus contortus in Ile de France and Santa Ines lambs.

The effect of Haemonchus contortus infection in sheep fed with a moderate and high protein content diet was evaluated in two breeds of sheep. Forty-eight Ile de France and Santa Ines lambs were maintained indoors since birth, in worm-free conditions. The lambs were allocated after weaning in four groups of six animals per breed, which were either infected or remain uninfected and given access to either a moderately or highly metabolizable protein diet. The moderately and highly metabolizable protein diets were calculated to supply 75 and 129 g metabolizable protein per kg of dry matter (MP/kg DM), respectively. The infection consisted of a trickle infection with 300 infective larvae, three times a week, for 12 weeks. Significant differences were observed for mast cell, globule leukocyte and eosinophil counts in the abomasal mucosa of the infected groups compared to the control of both breeds (P<0.05), regardless of the diet supplied. Significantly higher IgA anti-L5 antibody was detected in the infected Santa Ines groups than in the infected Ile de France groups (P<0.05). Increased metabolizable protein supply resulted in larger body weight gain and higher packed cell volumes for both breeds (P<0.05). Both breeds showed an increased ability to withstand the pathophysiological effects of H. contortus infection when given access to the highly metabolizable protein diet. However, increased metabolizable protein supply resulted in reduced worm burdens in Santa Ines lambs but not in the Ile de France lambs (P<0.05). The present results show that the increase in protein content in growing lamb diets may benefit resistance and resilience to gastrointestinal parasites but that these benefits may vary among breeds.

Relationship of abomasal histology and parasite-specific immunoglobulin A with the resistance to Haemonchus contortus infection in three breeds of sheep.

This study was carried out to evaluate the relationship of abomasal inflammatory cells and parasite-specific immunoglobulin A (IgA) in mucus, with the resistance to Haemonchus contortus infection in three breeds of sheep naturally infected with gastrointestinal nematodes. The breeds were the native Santa Ines sheep, and the European Suffolk and Ile de France breeds. Mast cells, eosinophils and globule leucocytes were enumerated in abomasal mucosa. Eosinophils within the sub-mucosa also were counted separately. Histamine concentration was estimated in abomasal tissue samples. Enzyme-linked immunosorbent assay was carried out in mucus samples to determine the level of IgA anti-H. contortus third and fifth instar. There were no significant differences among group means of these variables (P>0.05). The correlation coefficients between fecal egg counts (FEC)xmast cells (r=-0.490; P<0.05) and FECxeosinophils in sub-mucosa (r=-0.714; P<0.01) was significant in the Santa Ines sheep. In the Ile de France group, the correlation coefficients between globule leucocytesxFEC (r=-0.879; P<0.001) and histaminexworm burden (r=-0.833; P<0.01) were also significant. In the Santa Ines and Ile de France sheep, correlation coefficients between IgA anti-L3xworm burden and IgA anti-L3xFEC were negative. In general, inflammatory cells and IgA-parasite-specific in abomasum were inversely associated with H. contortus worm burden and FEC indicating that they may impair parasite development or fecundity in the three breeds of sheep. However, similar mean values of inflammatory cells and IgA were found in the resistant (Santa Ines) and in the susceptible (Suffolk and Ile de France) breeds of sheep. The enumeration of cells by histological assessment does not provide information on their functional activity, which may be different among breeds. Thus, the effect of breed on the functional activity of these and other inflammatory cells is an important area for further study.

Resistance of Santa Ines, Suffolk and Ile de France sheep to naturally acquired gastrointestinal nematode infections.

A study was conducted to assess the breed resistance against nematode infections in Santa Ines, Ile de France and Suffolk male lambs over a 9-month period in São Paulo state, Brazil. Lambs were born during the winter (year 2000) and were weaned at 2 months of age. The animals were then housed and treated with anthelmintics to eliminate natural infections by gastrointestinal nematodes. In late October 2000, lambs were placed in a paddock, where they stayed until August of the following year. Fecal and blood samples were taken from each animal every 2 weeks. On the same day, a pasture sample was collected to determine the number of infective larvae on the herbage. To prevent deaths, individual treatment with anthelmintics was provided to lambs with fecal egg counts (FEC) higher than 4000 eggs per gram (EPG) or with a packed cell volume (PCV) lower than 21%. In August 2001, all animals were slaughtered and the worms present in samples of the gastrointestinal contents were identified and counted. Most of the Suffolk and Ile de France sheep received three to six anthelmintic treatments over a period of 7 months, while most of the Santa Ines were not treated. Reductions in PCV and plasma protein values associated with high FEC and worm burdens were recorded, particularly, in Suffolk and Ile de France lambs. Haemonchus contortus and Oesophagostomum columbianum burdens and number of nodular lesions caused in the large intestine by O. columbianum larvae were significantly lower in Santa Ines sheep. All three breeds showed similar Trichostrongylus colubriformis worm burdens. The relative resistance of Santa Ines young male sheep was superior to that of Suffolk and Ile de France sheep.

Response of Corriedale and Crioula Lanada sheep to artificial primary infection with Haemonchus contortus.

Clinical, parasitological and biochemical parameters were evaluated in Corriedale and Crioula Lanada sheep after a single experimental infection with Haemonchus contortus. Ten 4-month-old worm-free lambs, of each breed, were infected with 200 L3 H. contortus per kg live weight and four uninfected animals of each breed were used as controls. Every week, the animals were weighed and blood and faecal samples were collected for measurement of packed cell volume (PCV), total serum protein (TSP) and albumin (ALB), and the number of eggs per gram of faeces (EPG), respectively. Twelve weeks after infection, the animals were slaughtered. The worm burden was determined and samples of the abomasal mucosa were processed for determination of the number of eosinophils, mast cells and globule leukocytes. No significant differences in PCV, TSP, ALB, parasite burden or the cell populations of the abomasal mucosa were observed between breeds, but Crioula lambs had a lower EPG count. The comparison of the infected groups with their respective controls revealed significant alterations in PCV, TSP and ALB in the Corriedale lambs and in PCV, TSP, ALB and the density of eosinophils and mast cells in the Crioula lambs.