Flow cytometric testing of immunological effects of a phytomedicinal combination (equimun) and its compounds on bovine leucocytes.

One of the major goals of this study was to establish fast, reliable and sensitive assays for the quality control of immunomodulating phytopreparations and to determine whether pharmacological compounds or phytopreparations have effects on bovine immune cells. Flow cytometric methods were chosen because they are very sensitive in the detection of even subtle effects on cells. In this study, we addressed the question of whether these methods are useful in monitoring the effects of EquiMun and its compounds on bovine leucocytes in vitro. EquiMun is a fixed combination of Echinacea purpurea (Ec), Thuja occidentalis (Th) and elemental phosphorus (Ph) in different starting concentrations. Separated blood mononuclear cells (MNC) and polymorphonuclear cells (PMN, mainly neutrophils) were cultured for up to 44 h in vitro in the presence or absence of the tested substances. Whereas MNC were not affected by any of the compounds, EquiMun, Ec, Th and Ph significantly reduced the forward scatter (size) of cultured PMN without affecting their side scatter (granularity). The size effects were paralleled by a significantly enhanced viability of PMN after 20 h in culture. The observed effects were constant over wide concentration ranges and indicate a very similar reaction of leucocytes from individual cows. Whereas spontaneous generation of reactive oxygen species (ROS) by neutrophils was up-regulated by Ph and EquiMun, EquiMun down-regulated the phorbol ester-stimulated ROS production. However, ROS generation by neutrophils displayed a large inter-individual variation with less apparent, down-regulatory effects of EquiMun. The ability of PMN to kill target cells via antibody-independent cellular cytotoxicity showed small inter-individual variations and was enhanced by Ec and Th but not by Ph and EquiMun, probably due to dose-dependent effects. In summary, the flow cytometric characterization of cellular viability and shape changes of neutrophils seem to be a suitable and reliable approach for the quality test of immunomodulating phytomedicines based on bioassays.

Uptake of colostral leukocytes in the intestinal tract of newborn calves.

The role of colostral immunoglobulins for the protection of newborn calves has been studied extensively, but little is known about the importance of colostral leukocytes. To study the uptake of colostral leukocytes in the intestine of calves and to determine preferential sites for this uptake, FITC-labelled colostral cells derived from the respective dams were injected into intestinal loops with/without Peyer's patches of three male Holstein Frisian calves about 5h post natum. In adjacent loops, PBS was injected as control. Loops were excised after an exposure of 1.5-2h. FITC-labelled material and cells were detected by the direct immunoperoxidase method in paraplast sections. Twenty-five consecutive sections were evaluated from each localization. Uptake of labelled material and cells was observed in all three calves in the jejunal Peyer's patch and in two calves in the ileal Peyer's patch as well. In the jejunal Peyer's patch, labelled material and cells were present in epithelium, domes and sinuses around lymphoid follicles, whereas in the ileal Peyer's patch, they were found in the sinuses only. These findings confirm that uptake of colostral leukocytes through the intestinal barrier is possible and that the preferential route of uptake is through follicle-associated epithelium of Peyer's patches.

[Use of a Coffea arabica tosta extract for the prevention and therapy of polyfactorial infectious diseases in newborn calves]. / Einsatz eines Coffea arabica tosta Extrakts zur Prophylaxe und Therapie polyfaktorieller Infektionskrankheiten neugeborener Kälber.

Two studies have been carried out to evaluate the prophylactic and therapeutical effect of a 30%-extract from the coffee-bean seeds Coffeae arabicae on infectious diseases in newborn calves. 1. Within a large cattle-herd, which endemically showed a high proportion of infections within the gastroenteric and/or respiratory systems in calves, a randomised placebo-controlled double-blind study has been done. 50 newborn calves were given a subcutaneous injection of 10 ml Coffea-preparation 30% on first and third day of life. Another 50 calves received physiological saline as control. An index was set up which allowed to daily evaluate and compare body-temperature, consistency of feces, exsiccation-degree and breathing-rate of the animals. Besides this the number of therapeutical interventions and the number of days with disease-symptoms were recorded. Calves treated with Coffea-extract showed: on first and second day of life less animals with body-temperature below physiological values (p < 0.001 or 0.1 resp.), during the first period of diarrhea (between fourth and sixth day) significantly lower tendency of diarrhea (p < 0.1; 0.001; 0.005 resp.), after the second period of diarrhea (around the 9th day of life) a better and quicker recovery and a lower tendency of exsiccation (p < 0.05 on day 10 and 11) as the control-calves. Besides this the average duration of illness was shorter (4.7 instead of 7 days) and the average number of therapeutical interventions were less (3.1 instead of 4.5) than in control-calves. 2. Within four cattle-herds endemically showing a high rate of diarrhea in newborn calves the morbidity in a total of 371 animals could be dropped from about 45% to 10% by prophylactic administration of one to three s.-c.-injections of 10 ml Coffea-preparation together with one or two million i.U.Vit.A. one time perorally. For prophylactic use two injections of coffea preparation. on day 1 and 4 of life proved to be efficient under the given circumstances. Therapeutically the daily administration of a combination of Coffea-extract together with oral drugs containing tannic substances and diet-feed could reduce the mortality in animals with acute disease to about 30%.

The influence of colostral leukocytes on the course of an experimental Escherichia coli infection and serum antibodies in neonatal calves.

Three hours post natum, 20 calves were orally infected with 10(9) colony forming units of an enteropathogenic E. coli. Immediately after infection and on 2 days subsequent, ten of the calves (COL-) received cell-deprived pooled colostrum and the other ten (COL+) pooled colostrum supplemented with colostral cells. The COL+ calves excreted significantly less bacteria of the infectious strain with their faeces in the first week after infection and reached the lower limit of detectability earlier than the COL- calves. Colostral leukocytes obtained from cows which developed clinical mastitis (n = 2), showed a marked reduction in the number of shed bacteria. The concentration of IgA and IgM specific antibodies against E. coli in the serum of the COL+ calves was significantly higher in the early postnatal period than in the serum of the COL- calves and remained at a slightly higher level throughout the whole investigative period. In addition to humoral substances of the colostrum, colostral leukocytes obviously contribute to the passive immunity and resistance of the newborn calf. The quality and quantity of the leukocytes seem to be of crucial importance to their efficiency.

The influence of colostral leukocytes on the immune system of the neonatal calf. IV. Effects on bactericidity, complement and interferon; synopsis.

The influence of colostral leukocytes on the bactericidity of whole blood of calves against a strain of E. coli and on the activities of haemolytic complement and interferon-alpha (the antiviral activity of sera resisting an acidic treatment at pH 2 for 6 h) in the serum was investigated during a period of 4 weeks using 4 experimental groups. The calves received either complete colostrum (COL+, n = 16), cell-depleted colostrum (COL-, n = 16), cell-supplemented milk substitute (MS+, n = 7) or pure milk substitute (MS-, n = 6) during their first three days of life. The bactericidity of whole blood of the COL+ group was significantly higher on the second and third days of life while the activity of haemolytic complement was lower after the first week as compared to the COL- group. No interferon-alpha was detectable in the sera of both COL groups. The bactericidity of the MS groups was significantly lower than that of the COL groups after the first day of life. It was significantly lower in the MS+ group after one week of life while the activity of haemolytic complement was higher than that of the MS- group. Three out of 5 MS- and only one out of 7 MS+ calves had low titres of interferon-alpha in their sera on the third day. Three out of 6 MS- calves died and 5 out of 7 MS+ animals. The mean day of death was 4.0 in the MS- and 8.4 in the MS+ group. Based on the in vitro results of this and the previous three communications it can be concluded that leukocytes which are an integral part of normal bovine colostrum, influence immunological reactions of the calf and that they may enhance its defence against infection. Colostral leukocytes in the absence of humoral components of the colostrum are not able to prevent fatal losses in the calves due to natural infection, although their influence on immune responses of the calves was detectable in vitro.

The influence of colostral leukocytes on the immune system of the neonatal calf. III. Effects on phagocytosis.

The influence of colostral leukocytes on the activity of phagocytic cells from the blood of calves, in particular the concentration of neutrophils (PMN) in blood, ingestion of Streptococcus agalactiae, reduction of NBT-dye and activity of lysozyme, was investigated for four weeks using four groups. The calves received either complete colostrum (COL+, n = 16), cell depleted colostrum (COL-, n = 16), cell-supplemented milk-substitute (MS+, n = 7) or pure milk-substitute (MS-, n = 6). Calves of the COL+ group had a significantly lower PMN concentration in their blood on day 2 and a significantly higher activity of lysozyme during their first three weeks of life as compared to the COL- animals. A postnatal increase in number of ingested Streptococcus agalactiae test bacteria per 100 phagocytic cells occurred later in the COL+ calves than in the COL-. No difference between both COL groups in NBT-reduction was observed. The calves of the MS+ group showed higher lysozyme activity and a retarded increase in the ingestion of test bacteria during the first week of life as compared to the MS-. The MS+ group had a transient neutrophilia on the second day of life while the concentration of PMN was not altered in the MS- from the first to the second day.

The influence of colostral leukocytes on the immune system of the neonatal calf. II. Effects on passive and active immunization.

The influence of colostral leukocytes on the concentration of immunoglobulins and antibodies against an enterotoxigenic strain of E. coli in the sera of newborn calves was investigated for four weeks using four experimental groups. The calves received either complete colostrum (COL-, n = 16), cell-supplemented milk substitute (MS+, n = 7) or pure milk substitute (MS-, n = 6) during the first three days of life. The cows were not specifically immunized. The sera of the COL+ calves had significantly higher concentrations of antibodies against E. coli mainly of IgG1 specificity on the second day of life as compared to those of the COL-. The sera of the COL+ calves contained significantly more IgM on days 2 and 5 and slightly more IgA during the first week. Both COL groups had equal concentrations of serum IgG. It appears that colostral leukocytes which are an integral part of the colostrum enhance the passive immunity of the neonatal calf, especially in regard of antibodies and immunoglobulin classes which are essential for intestinal immunity. The concentration of IgM in the sera of the MS+ calves was reduced, that of IgG did not rise to appreciable amounts; the IgA synthesis started one week later as compared to the MS- group. The administration of isolated colostral cells led to an impairment of the natural active immunization.

The influence of colostral leukocytes on the immune system of the neonatal calf. I. Effects on lymphocyte responses.

The influence of colostral leukocytes on lymphocyte counts in the blood of calves and on lymphocyte responses, in particular the Concanavalin A-induced blastogenic response in vitro and the formation of antibodies against sheep erythrocytes, was investigated for four weeks postnatum using four experimental groups. The calves received either complete colostrum (COL+, n = 16), cell-depleted colostrum (COL-, n = 16), colostral cell-supplemented milk substitute (MS+, n = 7) or pure milk substitute (MS-, n = 6) during their first three days of life. In contrast to the calves fed with cell-depleted colostrum (COL-) the calves fed with complete colostrum (COL+) showed no decrease of lymphocyte numbers in the blood on the second day of life, uniform blastogenic responses to two different Concanavalin A concentrations, slightly enhanced antibody formation against sheep erythrocytes and a high spontaneous proliferation of mononuclear cells during the first week of life. In the calves fed with milk-substitute supplemented with colostral cells (MS+) a higher blastogenic response to Concanavalin A and an intensified formation of antibodies against sheep erythrocytes was observed as compared to the MS- calves. A passage of vital colostral lymphocytes through the intestinal wall is postulated. They seem to stimulate and regulate the blastogenic response and enhance the T-helper cell-dependent formation of antibodies against sheep erythrocytes in calves.

[Review article: colostral leukocytes and their significance for the immune system of newborns]. / Ubersichtsreferat: Die Kolostralleukozyten und ihre Bedeutung für das Immunsystem des Neugeborenen.

The colostrum contains a comparably high concentration of leukocytes as the peripheral blood. The majority of them are vital leukocytes, namely neutrophils, macrophages and lymphocytes. There is some evidence in mouse and man that lymphocytes from the gut-associated lymphoid tissue home selectively to the peripartal mammary gland. The phagocytic cells may be involved in the transportation of certain immunoglobulins into the neonate. In vitro colostral leukocytes exhibit a variety of immunological activities such as blastogenesis after mitogenic and antigenic stimulation, cytotoxicity and phagocytosis, but the medium milk confines these activities in comparison with those of blood leukocytes. Intact colostral leukocytes reach the gut of the gut of the newborn and may even cross the intestinal wall, gaining access to the neonates system and influencing its immunologic reactions, e.g. hypersensitivity and antibody-formation. The knowledge on the significance of colostral leukocytes for the protection against infection of the neonate is still limited.