Zeolites Applications in Veterinary Medicine

Zeolites have a wide range of use, from construction industries, aquaculture industries, agriculture, space research to human and veterinary medicine. This broad application of natural and synthetic zeolites is given by their main properties: adsorption, molecular sieving and cation exchange capacity. In this chapter the main use of zeolites in veterinary medicine is reviewed. The beneficial effects of zeolites in animal nutrition, on mycotoxins, as an adjuvant in anticancer treatment and in increasing passive immunity of newborn ruminants are reported. Furthermore, multiple advantageous immune effects of zeolites such as their antioxidant capacity or their non-specific superantigen-like immunoglobulin action are also reviewed. Finally, their main positive effect on passive immunity in newborn calves is discussed. Literature data reviewed confirms their beneficial role in newborn calves during colostral period.


Introduction
The first group of zeolite minerals was discovered in 1765 by a Swedish mineralogist A.F. Cronstedt who described several species found in well-defined crystals. He noticed that some heated minerals began to lose their constituent water with a boiling-like appearance, hence the name of zeolite (from the Greek words "zeos" and "lithos" which translate as "boiling stone") [1].
Zeolites are natural, hydrated, crystalline aluminosilicates made up of threedimensional networks extended by AlO 4 and SiO 4 linked together by oxygen atoms, which make up a rigid, open, honeycomb-like skeleton, generally including cations which neutralize the excess negative charge of aluminum tetrahedra and water molecules. Each AlO 4 tetrahedral network supports a net negative charge that is balanced by a cation, usually from the I-A or II-A group (Ca, Mg, Na, K, Fe). These ions are not part of the zeolite network and can be changed by other cations such as heavy metals (Hg, Pb, Cd) or ammonium ions [2][3][4].
There are 67 natural zeolite minerals accepted by the Natural Zeolites Commission of the International Zeolite Association (IZA) and all have a unique three-letter code [5].
Clinoptilolite of sedimentary origin, generally the most used natural zeolite, is authorized by the Commission Implementing Regulation (EU) no. 651/2013 as a feed additive for all animal species [6]. In the United States, clinoptilolite belongs to the sodium aluminosilicate category and has the status of Generally Recognized as Safe (GRAS) (Code of Federal Regulations CFR, Title 21, Section 182.2727) [7].
Due to their main properties: adsorption, molecular sieving and cation exchange, zeolites have a wide use in many areas. For example, in agriculture, natural zeolites are used to obtain fertilizers capable of better nitrogen retention and in a slow and controlled release of fertilizers, nitrogen use efficiency (NUE) increase [8,9]. In aquaculture industry natural, synthetic or modified zeolites are used as adsorbents for ammonia removal from fish farming ponds and transportation tanks, as a cation-exchanger for removal of different toxic heavy metals from fresh water and sea water cultures and as a feed supplement to enhance fish growth [3]. Also, zeolites can increase the nutrient (by addition of micronutrients) and water use efficiency of drylands (by their water holding capacity) [9]. Natural zeolites are used in wastewater, surface waters, ground and underground water, drinking water treatment [10,11], in decontamination of radioactive waste water [12] and in agro-industrial wastewater treatment due to their exceptional cationic exchange and adsorption properties [13]. In construction field through their excellent properties, mainly porosity, specific weight and adsorption, they can be used as a building stone [14], in zeoponic substrates-artificial soils developed by the National Aeronautics and Space Administration-for plant growth in space [15], and as potential slow-release carriers for herbicides, insecticides and other organic compounds, protecting in this way the environment from chemicals [16].
In animal production, alternative products as zeolites are a solution to ensure health, productive performance (yield and quality of carcass, milk yield), to reduce the effects of mycotoxins on animal health status, to remove selectively pathogens from the animal gut without reducing microbial richness and finally to increase farm profitability. All of these effects has been extensively studied in the last decade [17][18][19][20][21][22][23][24][25][26][27][28] and are schematic represented in Figure 1.
Clinoptilolite is also used as a biomedical feed ingredient due to its beneficial properties as a growth-promoter and immunostimulant and can constitute an alternative to antibiotic growth promoters [29], since European Union legislation has banned the use of antibiotics for growth promotion in 2006, because the overuse of antibiotics in animals can contribute to emergence of antibiotic resistance [30].
Zeolites can have a protective effect in intoxication and in reducing parasite infestations. These effects are evidenced by researchers who observed that clinoptilolite (2 g/kg) could have some protective effect in organophosphorus poisoning in sheep by protecting the rumen flora and by preventing the decrease of cholinesterase activity [31]; in lead intoxication in mice, clinoptilolite given in 10/1 ratio  (clinoptilolite/Pb) adsorbed 91% of Pb, and when supplementing 3% clinoptilolite feed to pigs that received 150 ppm CdCl 2 , clinoptilolite prevented Cd-induced anemia by adsorption of Cd [32]; and in reducing the excretion of cysts in goat kids with giardiasis [33]. It also had beneficial effects in infestations with nematode larvae in lambs, producing an increase in feed consumption and in body mass [34].
Some types of zeolites are studied for their adsorption properties in order to improve the life of people suffering from chronic kidney disease, who need to undergo weekly hemodialysis. Dialysis membranes made from zeolite and polymer are studied in order to improve the performance of hemodialysis. Nanofiber membranes made from zeolite and polyacrylonitrile (PAN) adsorb creatinine, with the best results for 940-HOA (beta) zeolites (25,423 μg/g in 625 μmol/L creatinine solution) [35] and P87 zeolite in combination with polyethersulfone, used because of their improved resistance to fouling, thermal stability, chemical resistance and due to their high adsorption level of indoxyl sulfate (550 μg/g membrane) [36]. When zeolite was used along polyethersulfone (PES) and dimethyl formamide (DMF) in proper concentration: 17:0.5:82.5 (PES:zeolit:DMF), creatinine concentration decreased by 91.99%, which suggests the possibility of using these membranes in haemodialysis [37].

As dietary supplements in animal nutrition
In animal husbandry, natural and synthetic zeolites have been mostly used to improve productive performance. The proposed mechanisms involved in achieving the increase in productive performance in animals are: ammonia binding, reducing toxic effects of ammonia produced by intestinal microbial activity; low passage rate of digesta through the intestines and more efficient use of nutrients; enhanced pancreatic enzymes activity-favorable effect on feed components hydrolysis over a wider range of pH, improved energy and protein retention; elimination of mycotoxin growth inhibitory effects [32].
Due to the beneficial effects of the gradual release of ammonia ions on microbial synthesis in the rumen, zeolites are used especially in high non-protein nitrogen feed ratio. In vitro and in vivo experimental studies have shown that 15% of ruminal NH 4 + can be adsorbed by zeolites, thus reducing the toxic effects of urea (increased rumen pH and ammonia concentration in rumen and blood). Thus clinoptilolite (6%) in the feed of dairy cows receiving urea significantly reduced the concentration of NH 4 + in the rumen [32]. Also, a decrease in ruminal pH in diets with 1% clinoptiloliten is reported [38].
Milk fever and ketosis are the most common metabolic diseases that occur in cows with high milk production. Cows that received zeolite (1 kg zeolite/day for 4 weeks before calving) did not experience subclinical hypocalcemia [39]. Also, the administration of zeolite A (sodium aluminosilicate) to pregnant cows during the dry period (1.4 kg zeolite A/day in the last 2 weeks of gestation) reduced the incidence of milk fever. The mode of action of synthetic zeolite A is to reduce the bioavailability of fodder calcium at the gastrointestinal level (calcium binding capacity of zeolite is 110 mg/g Ca at pH 11), stimulating Ca-homeostatic mechanisms before calving. At calving, the plasma level of calcium was significantly higher in the experimental group (p < 0.0001); with a slight drop of inorganic magnesium and phosphorus, that set up a week postpartum [40].
When clinoptilolite was administered (2.5%) in the last month of gestation, the incidence of milk fever was 5.9%, compared to 38.9% in the control group. Also, clinoptilolite (2.5%) administered during the dry period reduced the incidence of ketosis (5.9%) by improving the energy metabolism through increased production of propionate in the rumen and by better recovery of feed [32]. Katsoulos et al. revealed that long-term clinoptilolite administration (from 4 weeks before calving to the next dry period) at different doses (1.25 and 2.5%) did not have adverse effects on the liver and serum glucose concentrations, ketone bodies, total protein and urea did not change, with a higher milk production and a lower ketosis incidence [41]. Moreover, other important minerals such as: Cu, Zn, Fe were not influenced by the long-term administration of clinoptilolite (1.25 and 2.5%), which highlights the safety of this natural compound [19].
Also, in combination with yeast, clinoptilolite (Rumencure: yeast 60% and clinoptilolite 40%) given to cows for a long period (30 days) had no apparent adverse effects on their liver function and on some biochemical parameters (glucose, ketone bodies, blood urea nitrogen and total proteins) [42].

Positive effects against mycotoxins
Due to the increased incidence of contamination with mycotoxins, it has been attempted to use inert feed adsorbents to bind mycotoxins, thus reducing their intestinal absorption and toxic effects on animals and animal products. Annually, it is estimated that about 25% of the world's harvested crops are contaminated by mycotoxins, leading to huge agricultural and industrial losses [43]. The first adsorbents successfully used in poultry, swine, sheep and bovine breeding were phyllosilicates, namely bentonite [32]. The adsorption process is strongly related to the pore size, the adsorbent contact surface, polarity, solubility, and the size of the mycotoxin molecules that are adsorbed (e.g., aflatoxins B1 and B2 have 5.18 Å and aflatoxins G1 and G2 are 6.50 Å). Clinoptilolite has the highest in vitro adsorption, over 80% for aflatoxins B1 and G2 [44], with effects demonstrated especially in the poultry industry [45][46][47][48][49], but also in piglets [22], lambs [17] and dairy cattle [50] as are presented in Table 1.

Adjuvant in anticancer treatment
The first studies of anticancer effect of zeolites were performed at the beginning of 2001 when it was observed that the treatment with clinoptilolite of different tumors in mice and dogs have improved their life span and tumors have decreased in size [54]. In vitro studies using cancer cell cultures revealed the clinoptilolite inhibitory effect on protein kinase B (B-Akt), which reduced the growth of cancer cells and increased their apoptosis. Inhibition occurred only in the presence of serum. This finding suggests that adsorption of serum components may be a possible mode of action. Adsorption of molecules involved in transduction signals, such as inositol, phosphatides and Ca, can contribute to its therapeutic efficacy. It also induces expression of tumor suppressor proteins, p21 WAF1/CIP and p27 KIP1, blocking the growth of cancer cell lines. It is assumed that clinoptilolite reduces the exchange rate of intestinal epithelial cells, prolonging their activity, and that silicates and aluminosilicates can interact directly with some cells by modifying their intracellular pathways, and this leads to the alterations in the regulation of gene expression. Changing the order of interaction of other proteins with membrane proteins may be involved, since membrane transport is required to activate protein kinase B [54]. Studies in mice injected i.v. with melanoma cells but receiving a micronized zeolite through gastric intubation for 28 days, revealed an increase in allogeneic graft versus host (GVH) in lymphocytes in the lymph nodes and a reduction in pulmonary metastases. The researchers' hypothesis is that the local inflammation caused by zeolite application, attracts peritoneal macrophages, and these cells in turn produce TNFα that stimulates spleen T-cells, which amplify the local inflammatory response [55]. Also, a reduction in the metabolic rate of cancer cells and a reduced production of 4-hydroxyinonenal following an anti-cancer treatment (Doxorubicin) along with tribomechanically micronized clinoptilolite, having in this way a potentiator effect on anticancer drugs, were reported [56].
In another in vitro study done on mouse fibrosarcoma cells and other types of cells incubated for 24 h together with clinoptilolite researchers observed that the number of viable cells, DNA synthesis and activity of EGF-R, PKB/Akt and NFKB was reduced while apoptosis was enhanced maybe because clinoptilolite affects cellular microenvironment through mechanisms that are dependent on its characteristics [57].

Effects on health status and growth performance
Clinoptilolite is also used as feed ingredient due to its beneficial properties as immunostimulant. One explanation of beneficial immune effects of silica, silicates and aluminosilicates could be their action as non-specific superantigen-like immunoglobulins (SAg). SAg are viral and bacterial toxins that are capable of activating a large population of T-cells. Activation occurs as a result of the simultaneous interaction between SAg, the T cell receptor (TcR) variable region β and the major histocompatibility complex (MHC) class II molecules on the surface of antigen presenting cells (APC). Consequently, SAg stimulates 10-30% of T-cells, as opposed to 0.01-0.0001% as it stimulates common antigens. Proinflammatory macrophages belonging to APC cells, CMH class II are activated by the particles of silicates [58].
An indirect action of clinoptilolite on the immune system is also achieved by its antioxidant capacity. Sverko et al. showed that administration of tribomechanically-activated clinoptilolite (12.5%) alone or together with Urtica dioica extract in mice per os for 3 weeks significantly reduced lipid peroxidation processes in the liver and significantly increased the content of superoxide-dismutase, an antioxidant enzyme. The antioxidant role of clinoptilolite is probably given by positive electrons that neutralize free radicals [59].
In weaned piglets that received 0.5% clinoptilolite for 5 weeks, it was observed that clinoptilolite was effective as an immunomodulatory agent by promoting the recruitment of circulating and intestinal immune cell subsets, even though it did not improve growth in weaned pigs, and generally failed to improve their feed conversion efficiency [60]; in heifers vaccinated against Escherichia coli (day 210 and 240 of gestation) the potentiating effect of clinoptilolite on the immune response was highlighted, and it indirectly improved the protection of calves [61].
Colostral period represents an important moment for the newborn ruminants, because in these species acquiring passive immunity is achieved exclusively through ingestion and absorption of adequate amounts of colostral immunoglobulins (Ig) [65]. Obtaining a good protection against neonatal diseases depends on how well this period is managed.
When we analyzed the effects of clinoptilolite supplemented in colostrum on blood serum protein electrophoretic pattern of newborn calves that received colostrum supplemented with 0.5 and 2% clinoptilolite, at 30 h after birth, the concentrations of γ-globulins, β-globulin and total protein in the group of calves that received 0.5% clinoptilolite (E1) were higher than in the control group by 42.11% (p < 0.05), 28.48% (p > 0.05) and 18.52% (p > 0.05), respectively, and were higher, but not significantly, in the group that received 0.5% clinoptilolite (E2), and a significantly lower albumin/globulin ratio in groups E1 and E2 (29.35%, p < 0.05 DOI: http://dx.doi.org/10.5772/intechopen.87969 and 35.87%, p < 0.05, respectively) was reported, compared with the control group at 30 h postpartum, which indicates an obvious increase in globulin fractions in experimental groups. Clinoptilolite was effective in improving passive transfer in newborn calves, better if added in colostrum in a dose of 0.5% than in a dose of 2% (Figure 2) [67].
A possible way of clinoptilolite action is explained, based on observation in Wistar rats that received zeolite for 34 days (6% of their weight) [68]. Some modifications of intestinal cells were observed, such as: the microvilli length was higher (1.2 vs. 2.0), the number of microvilli per 10 μm was higher (54.4 vs. 64.8) their diameter was smaller (0.17 vs. 0.13) compared to the control group, and also that the cellular organisms of the enterocytes, the density of mitochondrial membranes and the number of attached ribosomes were higher, which indicates a rise in the adaptation processes of the cells [68].
In an experiment carried out in 20 newborn calves in order to observe the clinical effects of clinoptilolite added in colostrum (20 ml clinoptilolite/L colostrum) during the first three meals, we concluded that administration of zeolites appears to reduce the incidence of diarrhea because only two calves from experimental group had health problems, one had bronchopneumonia and the other digestive transit difficulties with symptoms appearing after 28 days (not in neonatal period) in comparison with the control group where all calves had diarrhea in the first 11 days of life. The other parameter, growth performance measured on day 0, 45 and 90 revealed that during the first 45 days body weight of calves treated with clinoptilolite was significantly higher compared with the group of calves receiving only colostrum (C) (p < 0.0058) (E1/C = +16.96%). This statistical difference at 45 days may be explained by the high number of calves from the control group suffering from health problems and this affected the daily gain. At 90 days, the difference between groups was not significant (p < 0.1035) (E2/C = +7.19%) [69,70]. Similar results were obtained by Step et al. who found that body weight and average daily gain did not differ between treatment groups (clinoptilolite dosage was 0.5 and 2%) [23]. More recently, Ural et al. observed increased total weight and mean daily gain in calves that receive clinoptilolite (1 or 2 g/kg) in colostrum at calving, 12 and 24 h [71].
In another study, the addition of 0.5 g and 1 g/kg body weight per day in colostrum and milk for 45 days reduced fecal score and its severity, probably by retarding effect of clinoptilolite on intestinal passage rate [21]. The activity of clinoptilolite on reducing signs of diarrhea could be caused by: alteration of metabolic acidosis through effects on osmotic pressure in the intestinal lumen; or through retention of the enterotoxigenic E. coli thus limiting its attachment to the intestinal cellmembrane receptors); and also due to water adsorption property of zeolites, the feces appear drier and more compact [32].
In human medicine there are studies that support the beneficial properties of purified natural clinoptilolite as an anti-diarrheic treatment [72]. More recent studies performed on aerobically trained subjects, who received for 12 weeks zeolite-clinoptilolite supplementation, highlighted the positive effects of zeolites on intestinal wall integrity. The results were based on decreased concentrations of zonulin, an intercellular tight junction modulator, improving in this way intestinal barrier integrity [73].
Also, clinoptilolite improve antioxidant capacity in broilers [74]; it is used as a feed additive in fish diets [75] and in turkey diets [76].
Analyzing the effects of clinoptilolite on mineral parameters in newborn Romanian Black and White calves that received 5 g/l (group E1) and 20 g/l (group E2) at parturition, and 12 and 24 h postpartum, we observed that clinoptilolite supplementation increased serum Ca (with 37.34% in group E1, with 21.42% in group E2 in comparison with the control group and with 13.11% in E1/E2 at 30 h postpartum), P (with 37.34% in group E1, with 21.42% in group E2 in comparison with the control group and with 13.11% in E1/E2 at 30 h postpartum), Mg concentration (increased in groups E1 in comparison with the control group (p < 0.003) and E2 (p < 0.009) at 30 h postpartum in neonatal calves) with the most spectacular increase in iron concentration (with +144.70% in group E1 (p < 0.0005) and with +126.16% in group E2 (p < 0.002) at 30 h postpartum) [64].
When analyzing the same parameters in other breed (Holstein) and other colostrum quantity (3 L), we observed that the most significant effect (p < 0.0006) was on serum iron concentrations in experimental (27.64 ± 3.78 μmol/l) vs. control group (8.93 ± 1.26 μmol/l) and it did not have negative effects on other biochemical parameters (Ca, P, Mg, GGT, ALAT, ASAT, ALP) after 48 h postpartum, one more time proving that morpho-functional processes that take place in the newborns, necessary for adapting to the new environment, were not affected by clinoptilolite [63]. A possible explanation of increased iron level could be that in duodenum and in the anterior part of jejunum, where iron absorption takes place, clinoptilolite influences iron absorption due to the ion exchange properties, altering in this way the pH or reducing intestinal transit of digesta, which could lead to a better utilization of nutrients [32,77]. It has been shown previously that low intestinal motility and acidic pH promote iron absorption and that in the bovine neonate, the pH of the whole intestinal content ranges from 5.5 to 6.5; also, the motility of the gastrointestinal tract becomes well organized only after 2-3 days of postnatal life [78,79]. This feature could be important in preventing iron deficiency anemia (Fe < 14.32 μmol/l) especially in veal calves fed exclusively with milk.
Short term supplementation of clinoptilolite did not affect hepatic and renal function of newborn calves and that morpho-functional changes of the newborn organism in adapting to extrauterine environment were normal, without any influence of clinoptilolite, as observed after analyzing α-amylase, total bilirubin, creatinine, uric acid, urea, glucose, cholesterol and triglycerides. Biochemical values were measured in the first 48 h in the newborn calves. Values recorded were physiological DOI: http://dx.doi.org /10.5772/intechopen.87969 for the neonatal period and had no significant difference between groups, highlighting once more the safety of clinoptilolite in newborn nutrition [80].
The activity of the most important enzymes is changing very fast after the first feedings. We observed that adding clinoptilolite in the first three meals of colostrum influenced the enzymes as follows: GGT activity significantly increased in group E2 (20 g/L clinoptilolite) at 6 h after birth (E2/C: +64.83%, p < 0.05) and in group E1 (5 g/L clinoptilolite) at 16 h after birth (E1/C: +118.55%, p < 0.05) in comparison with the control group (C-received only mother colostrum); ALP increased after birth in all calves and adding clinoptilolite in colostrum influenced activity of ALP only in group E1 at 30 h postpartum; transaminases were low at birth in all calves but after feeding they increased, this coincided with the period when a morphofunctional condition of the liver is changing in a newborn calf; adding clinoptilolite (5 g/L colostrum) to colostrum determined increased ASAT (E1/C: +71.58%, p < 0.01) and ALAT (E1/C: +278.82%, p < 0.006) activity at 30 h postpartum [81]. As literature data suggests, serum GGT is the only enzyme to increase markedly as a result of its absorption from the colostrum; other serum enzymes, such as aspartate aminotransferase (ASAT) and alkaline phosphatase (ALP), are presumably released from the tissues of the calf [82]. A good interpretation of the serum enzyme activity in newborn calves must consider the physiological increase which occurs after feeding colostrum in the first days after parturition, a period very important for the calf but also for the cow [83].

Conclusion
Based on our research data and on continuously published literature data worldwide regarding the use of zeolites in veterinary medicine, we confirm that they can be used in animal nutrition as feed additives, mainly to reduce the gastrointestinal absorption of mycotoxins; in newborn calves, they can be used as enhancers of passive immunity during colostral period; also, to increase health status and growth performance of animals and as an adjuvant in anticancer treatment, with a promising perspective in this field.

Author details
Marc Simona* † and Tulcan Camelia † Faculty of Veterinary Medicine, Banat's University of Agricultural Sciences and Veterinary Medicine "King Michael I of Romania" from Timişoara, Romania *Address all correspondence to: simo20_med@yahoo.com † Equal contribution authorship.
© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.