Herb as agents affecting the immunological status and growth of piglets weaned with body weight deficiency

Immune deficiency was found in weaned pigs suffering from runting-stunting syndrome. It was manifested as decreases in: percentage of T-lymphocytes, phagocytic activity of neutrophils, the number of neutrophils capable of phagocyting and reducing nitrotetrazolium blue (NBT), D T H skin reaction to l-chloro-3,4-dinitrobenzene (DNCB). The runt pigs were treated with: a mixture of 5 herbs (Chamomile flowers, Calendula florets, Fennel fruits, Fenugreek seeds, Sweet Basil herbs) and Biostymina an aqueous aloe extract, given separately or together with the herb mixture. Administration of the five-herb mixture and/or Biostymina resulted in enhancement of neutrophil activity. Restoration of the T-lymphocyte population was observed only in the group treated with both preparations. The 5-herb mixture was the most effective in promoting weight gain compen­ sation.


INTRODUCTION
The runting-stunting syndrome affecting suckling piglets develops into a serious production problem.The body weight deficiency at weaning later increases even two or three times during fattening as compared with healthy pigs of the same age (Rekiel, 1992).
Runting-stunting syndrome develops together with immune deficiency (Hennessy, 1987;Mysłowski, 1990;Świtała et al., 1994;Kołacz et al., 1994Kołacz et al., , 1995;;Bodak-Koszałka et al., 1996), hence the pigs affected with the syndrome are more likely to suffer from infectious diseases in their severe or chronic form.The immune response to the infectious agent is only partial.This increases the risk of the pathogens carrier introducing infections of an epizootiologic character.
It has been assumed in this work that administration of herb preparations enhancing the immune response and normalizing the digestive function, to pigs with body weight deficiency may promote restoration of the immune response and body weight compensation.
The aim of the study was to determine the immune response and growth after treatment with herb preparations of pigs that were underweight at weaning.

MATERIAL AND METHODS
The study was carried during out 42 days on a commercial pig farm.The piglets weaned at 28 th day of life were divided into four groups, with 12 runt pigs (4-5 kg body weight) in each.The body weight deficiency at weaning was a result of low body weight at birth and disorders in lactating sows.Litters in which diarrhea occurred prior to weaning were excluded from the study.The results of earlier studies show that colibacteriosis markedly reduces the immune response (Świtała et al., 1997).
The four study groups were denoted as G-C group (negative control), and 3 experimental groups: G-H, G-B and G-HB in which herb preparations were administered.In addition, the G-N group contained 12 normal pigs weighing 7-8 kg.
In group G-H, the five-herb mixture was composed of the following dry herbs (Herbapol, Poland): Chamomile flowers (Anthodium chamomillae), calendula florets (Flos calendulae), fennel fruits (Fructus foeniculi), Fenugreek seeds (Semen foenugraeci), Sweet basil herbs (Herba basilici) in equal parts by weight.During 42 consecutive days of the experiment, herbs in an amount of 1.5 g/kg BW were infused and diluted with water at a proportion of 1:20.A fresh portion was poured into plastic containers every day, to which the animals had ad libitum access.
In group G-B group, an aqueous aloe extract under the trade name of Biostymina (Herbapol, Poland) was administered orally at a dose of 4 ml on days 1, 14 and 12 of the experiment.
In group G-HB group, both the herbal decoction and Biostymina were administered as described for groups G-H and G-B.
Each group of pigs was kept in a separate pen with free access to water and feed (PP-prestarter mixtures).The feeding value of the PP-prestarter mixtures was: ME, 13.5 MJ/kg; dry matter, 90%; crude protein, 23.2%; crude fat, 2.8%; crude fibre, 3.3%.
On the last day of experiment (day 42) venous blood was collected for the following tests: leukogram; phagocytic activity of neutrophils ex vivo (a) with the use of the nitrotetrazolium blue (NBT) reduction test according to Park et al. (1968) and (b) with the use of the Staphylococcus epidermidis phagocytosis test; percentage of T-Lymphocytes forming E-rosettes according to Binns (1978); the percentage of B-lymphocytes forming EAC rosettes according to Bianco et al. (1970); the IgG level with the use of radial immunodiffusion (RID kit, The Binding Site, U.K.); delayed type hypersensitivity (DTH) reaction to 1-chloro-3,4-dinitrobenzene (DNCB) in a skin test.
In the test of S. epidermidis phagocytosis, freshly collected blood (0.5 ml) was mixed with a suspension (0.35 ml) of live bacteria (density 3xl0 8 cells/ml) opsonized previously with swine blood serum and incubated at 37°C for 30 min.The smears were stained using the MGG method and read under immersion.The percentage of phagocyting neutrophils and phagocyte index (the number of phagocytized bacteria per one phagocyting cell) were determined.
In skin test with DNCB 0.2 ml of 2% DNCB was used epidemically to sensitize the pigs on day 42 of the experiment.A skin reaction (under the ear) was evoked by the same dose of DNCB 14 days later and measured after 24 h.
The pigs were weighed twice, on the first and last day of the experiment.
The data obtained in the study were analysed statistically using a Student's t-test.Differences with P<0.05 were considered significant.

RESULTS
A comparison of the immune response of runt-untreated pigs with normal pigs is presented in Table 1.No marked differences in the percentage of blood neutrophils or lymphocytes in G-N and G-C pigs were found.However, a significant, 37% (P<0.05),decrease in NBT-positive cells and an 11% (P<0.05)decrease in neutrophils phagocyting S. epidermidis were observed in G-C pigs.
In G-C pigs the percentage of E-rosette forming T-lymphocytes was 32% lower (P<0.05),whereas the percentage of B-cells forming EAC-rosette was similar to that in G-N pigs.In the runt-untreated pigs (G-C) 20% lower (NS) level of IgG and 30% decreased DTH reaction in the skin test with DNCB (P<0.05) were found.

Response of runt pigs to herbaceous preparations
In comparison to G-C pigs, the following increase in neutrophils was observed in treated groups (Table 2): in G-H, 63% (P<0.05), in G-B, 77% (P<0.01), and in G-HB, 104% (P < 0.05).Besides, an increase in neutrophils phagocytic activity occurred in those groups (Figure 1).The percentage of NBT-positive cells increased in group G-H by 123%, in G-B by 156%, and in G-HB by 130% (P<0.05).The percentage of neutrophils phagocyting S. epidermidis increased in group G-H by 36%, in G-B by 33%, and in G-HB by 26% (P<0.05).The phagocytic index increased in group G-H by 12%, in G-B by 16% and in G-HB by 43% (P<0.05) which proves the synergetic action of the herbs mixture and Biostymina.
The population of lymphocytes in pigs was not significantly changed after treatment with herb preparations (Table 2).Administration of the herb decoction or aloe extract increased the percentage of B-cells forming EAC rosettes (G-H 39%, G-B 50%, P<0.001).However, in both G-H and G-B pigs  there was no significant change in T-subpopulation forming E-rosettes (Figure 2).The opposite picture was seen in group G-HB, in which a significant increase in the percentage of E-rosette forming cells of 61% (P<0.05),i.e., the level similar to that of G-N pigs, which occurred together with slight stimulation of EAC rosette formation.The IgG level in the blood of G-H, G-B and G-HB pigs was not markedly different from that of the G-C group.In G-H pigs the mean IgG level was the lowest, while in G-HB pigs it reached the highest value.
In treated pigs (G-H, G-B and G-HB) the delayed-type hypersensitivity reaction to DNCB was only slightly higher than in G-C group (NS).

Growth of pigs
The initial body weight of entire runt population (G-C, G-H, G-B, G-HB) averaged 4.69 kg and was 2.43 kg (34%) lower than the weight of C-N pigs (Table 3).On the last day of the experiment the difference between G-C and G-N was 5.3 kg (29.6%).
Average daily gain (ADG) of G-N pigs was 257 g, while that of G-C 186 g (P<0.01).The ADG of treated runt pigs was were as follows: G-H 232 g, G-B 201 g, G-HB 194 g.
On last day of the experiment the pigs taking the herbaceous decoction (G-H) showed the highest body weight of the runt pig groups (G-C, G-B, G-HB).The difference in body weight in 70-day-old pigs between G-C and G-H groups was 1.49 kg (P<0.05).It is worth noting that the difference in ADG between runt pigs fed on herbs (G-H) and normal animals (G-N) was negligible (25 g).In the remaining groups (G-B and G-HB) the final body weight was not markedly different from that of G-C pigs.

DISCUSSION
The results confirm, that when the body weight deficiency occurs in young pigs then immune deficiency occurs as well.It is expressed by decreased neutrophils phagocytic activity, a decrease in the number of T-lymphocytes and a decrease in DTH skin reaction to DNCB.These findings corroborate our earlier results (Świtała et al., 1994;Kołacz at al., 1994;Bodak-Koszałka et al., 1996).
As is generally known, runting-stunting syndrome develops in young pigs on the background of under-or malnutrition.The latter leads to alimentary hypoproteinemia causing a reduction in lymphatic organs, followed by following their degenerative structural and functional changes, finally impairing phagocyting cell activity and antibody synthesis (Rzedzicki and Kowalska, 1992).Immune deficiency results in numerous infections, in the course of which one can observe stimulation of T-suppressor or temporal restriction of T-helpers activity.This leads to a decrease in immune system reactivity, obviously exposing the pigs to infections of secondary or mixed type (Pejsak and Truszczyński, 1993).
The five-herb mixture administered in the present study (G-H) proved to be immunostimulating.It seems possible that substances in Calendula florets were active in potentiating the cellular response in pigs.This herb is known to enhance defensive reactions, which makes it especially useful in the treatment of neoplastic diseases (Ożarowski, 1982).Chamomile flowers containing azulenes stimulating phagocytosis may have been directly involved in immune reactivity.The herbs could indirectly affect hormone and vegetative systems.For instance, Chamomile influences the function of the thyroid gland (Ożarowski, 1982).A correlation between thyroid function and a thymus-dependent response has been documented (Ślebodziński, 1993).Moreover, some substances contained in herbs (oils, silicon, calcium-phosphorus compounds) may constitute the kind of natural adjiuvants which can significantly enhance the response to antigens (Truszczyński and Pejsak, 1993).
Obviously, the mode of action of compounds entering into the originally composed mixture cannot be only univocally attributed to a chosen group of chemical substances present in those herbs.It is a fact that some of the compounds found in Fenugreek seeds, Chamomile flowers and Sweet Basil herbs may act on intestinal nutrient absorption and regulate intestinale peristalsis, whereas compounds isolated from Calendula florets and Fennel fruits stimulate secretion within the alimentary tract.Similarly, through their bactericidal action, Chamomile flowers and Sweet Basil herbs inhibit excessive growth of intestinal microflora, thus counteracting inflammation.These herbs appear to be stimula ting regeneration processes on the intestinal membrane after past gastricintestinal diseases.
The nutritive value of the herbs was of no less importance, especially as the herbs it included Fenugreek seeds which contain, among others, 20% of protein, 6-10% lipids, 2-6% mineral salts, carbohydrates, as well as vitamins A and D 3 in amounts comparable to cod-liver oil (Muszyński, 1958;Ożarowski, 1982).A mixture of herbs containing substances which regulate digestion and nutrient absorption affected the growth of G-H pigs, which was finally not much different from that in G-N pigs.Gajewczyk and Akińcza (1988) reported better feed intake and a 12% increase in daily gain after addition of a post-extractive infusion from Calendula florets and Chamomile flowers, a by-product of the pharmaceutical industry.Other authors (Mazurczak et al., 1973;Gajęcki, 1988;Maciolek, 1992) have found a more or less spectacular influence of herbs on daily gain of pigs.A positive impact on growth was atributed to the improvement of feed flavour, hence its better intake, as well as to the improvement of health and vitality.It could also be supposed that some herbs have an anti-stress effect, probably due to their content of sedative substances.
An immunostimulating reaction was expected after administration of aloe extract (Biostymina), whose immunotropic effects have been well documented (Ożarowski, 1982;Furowicz, 1989;Furowicz et al., 1990).The increase in phagocytic activity of neutrophils in G-B pigs resulted from the known properties of this preparation, such as intensified pathogenic cell absorption and increased granulopoesis.In increase in the lymphocytes B subpopulation forming EAC rosettes was also observed in the G-B pigs, while an increase in the IgG level was not reported.
In G-B pigs body weight gains were not markedly changed.Other studies carried out on young pigs (11-12 weeks) suffering from runting syndrome proved that after three doses of Biostymina of 2 ml/kg BW per dose, daily gain worsened by 13% and feed intake decreased by about 10% in comparison to the untreated pigs (Rekiel, 1992).These results suggest an undesirable effect of Biostymina on the growth of young pigs.The reason for such a reaction is not clear.In calves (4-5 months old) suffering from a chronic form of Bronchopneumonia enzootica, the immunostimulating of Biostymina such as significant increase in phagocytic reactions and serum IgG level was accompanied by its stimulating influence on daily gain (Furowicz, 1989;Furowicz et al., 1990).
Administration of the herbs mixture and Biostymina (G-HB) significantly affected the immune response of runt pigs.Besides the stimulating effect on the percentage of neutrophils phagocyting S. epidermidis, synergism of action of preparations on the number of phagocytized bacteria by those cells was not reported when those preparations were administered separately.However, the number of B lymphocytes forming EAC rosettes did not rise.A worse weight gain than that in the group taking the herb mixture alone was found (G-H), which suggests that Biostymina overcomes the stimulating influence of herbs on gain and inhibits the growth of pigs.

CONCLUSIONS
Young pigs with underweight at weaning were proved to suffer from immune deficiency manifested as a decrease in neutrophil phagocytic activity, in the number of T-lymphocytes in peripheral blood and in the DTH reaction to DNBC.
Administration of a five-herb to the form of a decoction diluted with drinking water, or Biostymina, an aqueous aloe extract, or both preparations together to those runt pigs appeared to restore the immunei reaction.
The five-herbs mixture, probably through its regulating action on digestive processes, was the most effective for weight gain compensation.
Biostymina did not restore weight gain of runt piglets.

TABLE 2
Number of white blood cells (106/ml) in runt pigs untreated or treated with herbaceous preparation * -P<0.05

TABLE 3
Body weight, average daily gain (ADG) and survival rate in runt pigs untreated or treated with herb preparations, and in normal pigs *-P<0.05