On-line version ISSN 1678-4162
Arq. Bras. Med. Vet. Zootec. vol.52 n.5 Belo Horizonte Oct. 2000
Parasitological characteristics and tissue response in the abomasum of sheep infected with Haemonchus spp.
[Características parasitológicas e resposta tissular do abomaso em cordeiros infectados por Haemonchus spp.]
T.C.G. Oliveira-Sequeira1, A.F.T Amarante1, J.L. Sequeira2
1Instituto de Biociências da Unesp, Campus de Botucatu
18.618-000 Botucatu, SP
2Faculdade de Medicina Veterinária e Zootecnia, Unesp, Campus de Botucatu
Recebido para publicação, após modificações, em 16 de fevereiro de 2000.
A histopathological study was conducted on the abomasal mucosa of 40 Corriedale sheep exposed to natural infection with Haemonchus spp. The sheep were allowed to graze on contaminated pastures for 14 days and, after being housed for 28 days, they were slaughtered. Fecal samples were collected for fecal egg counts (FEC) and abomasum samples were obtained for histopathological examination and eosinophil, mast cell and globule leucocyte counts. The number of Haemonchus spp. present in the abomasum was estimated from a 10% aliquot of its content. The number of eggs per female was estimated for 10 Haemonchus spp. females collected from each animal. The following significant correlation coefficients were obtained between the characteristics analyzed: number of Haemonchus and FEC (r = 0.86), female length and number of eggs per female (r = 0.60), female length and FEC (r = 0.53), number of eosinophils and number of Haemonchus (r = 0.48), number of eosinophils and number of globule leucocytes (r = 0.54), number of mast cells and female length (r = -0.39), and number of mast cells and number of globule leucocyte (r = 0.34). The characteristics that showed the highest correlation with animal load was FEC.
Keywords: Sheep, Haemonchus, eosinophil, mast cell, globule leukocyte.
Realizou-se um estudo histopatológico da mucosa do abomaso de 40 cordeiros da raça Corriedale, expostos à infecção natural por Haemonchus spp. Os cordeiros foram colocados em pastagens contaminadas por 14 dias e após 28 dias de estabulação foram necropsiados. Por ocasião da necropsia, foram colhidas amostras de fezes para a realização da contagem de ovos por grama de fezes (OPG) e amostras do abomaso para exame histopatológico e contagem do número de eosinófilos, mastócitos e leucócitos globulares. O número de Haemonchus spp. presente no abomaso foi estimado a partir de uma alíquota de 10% do conteúdo. O número de ovos por fêmea foi estimado em 10 fêmeas de Haemonchus spp. colhidas de cada animal. Coeficientes de correlação significativos entre as características analisadas foram: número de Haemonchus e contagem de OPG (r = 0,86); comprimento de fêmeas e número de ovos por fêmea (r = 0,60); comprimento de fêmeas e contagem de OPG (r = 0,53); número de eosinófilos e número de Haemonchus (r = 0,48); número de eosinófilos e número de leucócitos globulares (r = 0,54); número de mastócitos e comprimento de fêmeas (r = -0,39) e número de mastócitos e número de leucócitos globulares (r = 0,34). Das características analisadas a que apresentou maior correlação com a carga parasitária dos animais foi a contagem de OPG.
Palavras-Chave: Ovino, Haemonchus, eosinófilo, mastócito, leucócito globular
Several studies have been conducted to elucidate the mechanisms by which the hosts develop resistance to gastrointestinal parasites. Tissue eosinophil, mast cell and globule leucocyte populations have received special attention as indicators of the immune response since they are frequently associated with gastrointestinal parasitic diseases (Backer et al., 1993). Several investigators consider that the increase in the numbers of these cells in the mucosa are a strong indicator of their functional importance in the immune response associated with resistance to parasitism (Rotwell, 1989).
With respect to Haemonchus spp., the response of the host may have an unfavorable effect on the establishment and maintenance of the parasite at two different times during the cycle. In the histotrophic larval phase the phenomena of self-cure and rapid expulsion are protective mechanisms known to occur in sheep (Soulsby, 1987) and have both been associated with eosinophil and mast cell infiltration of the mucosa (Miller et al., 1983; 1985). Changes in female fecundity have been related to abomasum pH (Honde & Bueno, 1982), to infecting larval intake (Roberts & Swan, 1981), and to the intensity and duration of infection (Coyne et al., 1991).
In a study on rabbits infected with Trichostrongylus colubriformis, Mallet & Hoste (1995) did not detect a correlation between number of mast cells and number of worms in the intestine, but did detect a correlation between increased numbers of mast cells and reduction of egg production by the worms.
Gastrointestinal parasitoses represent the major sanitary problem for the sheep-raising industry in the State of São Paulo. The use of improved pastures in most rearing units, although allowing the exploitation of unit area by a larger number of animals, is a determinant factor of increased contamination and, consequently, of high infections. In coproparasitologic surveys, up to 100% prevalence of Haemonchus spp. has been observed in sheep (Amarante, 1995). This high prevalence, in association with the high pathogenicity of the agent, has caused Haemonchus spp. to be one of the major sheep parasites in the State of São Paulo.
The effect of breed on resistance to gastrointestinal nematodes is a well known fact, but the causes of these differences are still under intensive investigation. Even though the differences in susceptibility are also substantial among animals of the same breed (Wakelin, 1995), this is an area that still requires investigation. The present study was designed to determine the association between the tissue response of the abomasum especially in terms of eosinophils, mast cells and globule leucocytes and parasite burden, Haemonchus spp. fecundity (number of eggs per female) and faecal egg counts (FEC) in Corriedale sheep submitted to natural infection.
MATERIALS AND METHODS
The paddocks and animals used in the present study have been described in detail in a previous report (Amarante et al., 1997). Briefly, three paddocks similarly covered with Cynodon dactilon in terms of topography and watering. They had been exclusively grazed by sheep for the past few years.
The paddocks remained unoccupied by animals for a period of 57 days before the beginning of the experiment. After this time, five calves were placed in a paddock of 1 hectare (paddock 1), five calves and 16 sheep were placed in a second paddock of 2 hectares (paddock 2), and 16 sheep were placed in a third paddock of 1 hectare (paddock 3). The animals remained in these paddocks until the end of the experiment.
Seventy-two Corriedale sheep, aged four to eight months, were held in pens and treated with different antihelminthic classes in order to eliminate the natural infections they presented. After at least 10 days from the last treatment, two animals were placed monthly in each of the three paddocks described above. The sheep were kept in the paddocks for 14 days, after which they were housed again for 28 days. At the end of this period the animals were slaughtered.
The abomasum was separated and opened along the greater curvature. The content was removed and a 10% aliquot was fixed in 5% formalin and stored in plastic flasks. The abomasum was submitted to digestion with 1% hydrochloric acid (Ueno & Gonçalves, 1994) and the nematodes obtained were identified (Reineck, 1983; Ueno & Gonçalves, 1994) and counted.
The Haemonchus spp. parasite burden was calculated by multiplying by 10 the number of nematodes detected in the 10% aliquot and by summing this value to the number of Haemonchus spp. obtained in abomasum digestion.
A fecal sample was obtained from each animal for the quantitative diagnosis of gastrointestinal nematodes by the technique of Gordon & Whitlock (1939), modified. Another sample was obtained for fecal culture for the genus identification of third instar larvae by the method of Keith (1953).
After the abomasum has been opened and before the removal of nematodes, fragments of the organ were collected for histological analysis. One fragment was fixed for 24 hours in 10% neutral buffered formalin, routinely processed for histology and embedded in paraffin. Another fragment was fixed for 24 hours in Karnowsky solution (4% paraformaldehyde and 1% glutaraldehyde in phosphate buffer, pH 7.2) and embedded in glycol metacrylate (Polyscience).
After the worms present in the abomasum have been identified, samples were selected from animals that only presented Haemonchus spp. in the abomasum and also samples from animals that presented no worms in the abomasum. Thus, samples were processed from 35 animals parasitized with Haemonchus spp. and from four non-infected animals who grazed on pasture from July 1992 to April 1993.
The paraffin-embedded samples were cut into 5-7m m sections with a steel knife microtome which were stained with hematoxylin-eosin (HE). The metacrylate-embedded samples were cut into 1m m sections with a glass knife microtome which were stained with Giemsa.
The sections stained with HE were used for the description of the lesions provoked by the nematodes, and the sections stained with Giemsa were used for mast cell and eosinophil counts. Globule leucocyte counts were performed on sections stained with HE.
Eosinophil and mast cell counts were performed using a kpl 8x ocular micrometer with a 100 point grid and a 40x objective. Thus, the area delimited by the grid was 0.06mm2. Globule leucocytes were counted under a microscope fitted with a mercury vapor lamp using a 100x objective. In this case the area delimited by the grid was 0.01mm2. The cells were counted on 20 fields from the muscularis mucosa to the mucosal surface (Huntley et al., 1992).
Ten Haemonchus females from each parasitized animal were stored in neutral 5% buffered formalin, measured for length and ruptured in 2ml 0.9% saline solution. The number of eggs per female was calculated by counting the eggs in 400ml of this suspension.
The parasitological and histological data obtained were analyzed statistically by calculating the Spearman correlation coefficient.
The parameters obtained for the animals in each paddock were submitted to analysis of variance and the same analysis was used to determine the influence of season of the year.
All statistical analyses were performed using the Minitab version 11 software.
The secretory epithelium was intact in most animals, and the only glandular change observed was dilatation, although of moderate degree. Inflammatory alterations were observed in all animals and were characterized by diffuse (27 animals) or focal (13 animals) infiltrates involving the lamina propria and in some cases reaching the submucosa. These infiltrates consisted mainly of mononuclear cells and, to a lesser extent, of eosinophils. In the animals with no worms in the abomasum the infiltrate was exclusively mononuclear.
Vascular changes were limited to interstitial edema and, in some cases, to small focal points of hemorrhage.
The number of Haemonchus parasites detected ranged from 12 to 8,943, and FEC ranged from zero to 21,600. Four animals were not parasitized with Haemonchus at the end of 28 days of confinement.
Female length, calculated as the mean for 10 females from each parasitized animal, ranged from 13.7 to 23.3mm and the number of eggs per female ranged from 210 to 1590 eggs.
The number of eosinophils, mast cells and globule leucocytes ranged from zero to 230, from 16.7 to 266.7 and from 1 to 53 cells/mm2 of mucosa, respectively.
Correlation coefficients between parasite load, female length, FEC, number of eggs per female, number of eosinophils, mast cells, and globule leucocytes are presented in Table 1.
|Table 1. Correlation coeficients between parasitological results and cellular responses in the abomasal mucosa of 40 Corriedale lambs infected with Haemonchus spp.|
*P<0.05; **P<0.01; ***P<0.001
The fact that the animals were placed in different paddocks did not interfere with any of the parasitological or histological parameters analyzed (P>0.05).
The more relevant histopathological changes observed in the abomasal mucosa were glandular dilatation, mucosal edema and a predominantly mononuclear infiltrate. Similar changes have been reported previously (Salman & Duncan, 1984).
Eosinophils have been considered by several investigators to be important elements in the inflammatory response against parasitic helminths (Buddle et al., 1992) and eosinophilia, both in blood and tissue, has been frequently associated with the expression of a greater resistance to nematodes (Rotwell et al., 1988; Dawkins et al., 1989). However, conflicting results have been obtained in the evaluation of the association between eosinophilia and the manifestation of resistance to nematodes.
Douch & Morum (1993) reported an increased number of eosinophils in sheep immunized against T. colubriformis and later Douch et al. (1996) found a similar number of eosinophils in immunized sheep and in controls. Dineen et al. (1978) detected a positive correlation between number of eosinophils and parasite burden in sheep infected with T. colubriformis and later observed (Dineen & Windon, 1980) a weak negative correlation between these same characteristics.
The present results indicate the occurrence of a positive correlation between number of eosinophils and parasite burden (r = 0.47). Furthermore, these cells were found only in the mucosa of animals with worms in the abomasum. Comparable results were obtained by Salman & Duncan (1984). They observed that the number of eosinophils was higher in adult sheep than in lambs experimentally infected with L3 doses of Haemonchus calculated by weight and attributed this fact not to a greater expression of resistance but to the higher number of L3 received by sheep.
Helminth antigens are potent inducers of reagins or homocytotropic IgG (Jain, 1986) and consequently eosinophilia is expected to be proportional to the degree of stimulation by the parasite or to the parasite burden.
In the present study in which eosinophils were quantitated after the animals had held in pens for 28 days, clearly indicate that after the worms reach the adult stage the presence and number of eosinophils depend on the presence and number of worms in the lumen of the abomasum. This does not exclude the possibility that during the early phases of parasitism these cells may play a more relevant role in protecting the animals against the development of larval stages and in phenomena of rapid expulsion.
With respect to mucosal mast cells (MMC), several investigators have also been obtained conflicting results. While some studies suggest that mast cells may be activated during nematode rejection in sheep (Huntley et al., 1987; Emery et al., 1993), others demonstrate that sheep may express a high degree of resistance with no increase in MMC (Huntley et al., 1992).
According to Huntley et al. (1992), the proliferation and differentiation of mucosal mast cells require continuous or repeated stimulation by parasite antigens and the presence of large numbers of MMC is not a prerequisite for helminth elimination.
In the present study, whose objective was to determine the role of these cells in the phase in which the worms have already reached adulthood, there was a low correlation between MMC number and parasite burden (r = 0.13), but a significant correlation between MMC number and female length (r = 0.39).
Frequently the response of the host to helminths is investigated in terms of its correlation with parasite expulsion, with rare consideration of the fact that this response may interfere with the physiological functions of the parasite. Physiological alterations include reduced fertility, limited growth and morphological or structural damage (Krupp, 1961). Mallet & Hoste (1995) did not observe a correlation between MMC and parasite load in rabbits infected with T. colubriformis, but observed a negative correlation between mast cell number and egg production by the worms.
As is the case for eosinophils and mast cells, the association between number of globule leucocytes and manifestation of resistance to gastrointestinal nematodes has been reported in some studies (Bisset et al., 1996; Douch et al., 1996) but not in others (Patterson et al., 1996; Amarante et al., 1999).
According to Douch et al. (1996), the FEC is the characteristic that more directly reflects the parasitological status of sheep and is highly correlated with parasite burden, especially in young animals. The present results confirm this observation since the correlation between FEC and Haemonchus burden was 0.86. Furthermore, positive correlations were observed between FEC and female length (0.50) and between FEC and number of eggs per female (0.50).
The present results and those reported in the literature confirm the fact that the immune response against gastrointestinal parasites is quite complex since, in addition to the fact that each stage may present a varied repertory of antigens capable of inducing defined responses against each one (Miller, 1984), the contribution of each component of the immune response may vary, without necessarily interfering with the overall effectiveness of the response (Wakelin, 1995). In other words, it is unlikely that only one of the variables involved in the immune response can be used for predicting resistance.
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