Prevention of Mycotoxicosis in Poultry

Prevention of Mycotoxicosis in Poultry

Aflatoxin B1 Ochratoxin A Fumonisin B1 Trichothecenes: T-2 DAS DON

Aflatoxins are mainly produced by Aspergillus flavus and Aspergillus parasiticus. The most toxic is Aflatoxin B1. Aflatoxins are found as natural contaminants in cereals (specifically in corn, wheat, and rice) and cereal by-products and oilseed meals.

Liver symptoms: Hepatic lesions Fatty liver Increase of liver size Egg production symptoms Decreased egg production Soft egg shells Poor protein and aminoacid digestion Poor growth rate Mortality T1- Control, T2 3ppm of Aflatoxin

Research on the preventive effects of modified clinoptilolite Minazel Plus on MSc Radmila Resanovic* PhD Thesis Aflatoxin exposed poultry * University of Belgrade, Faculty of Veterinary Medicine Belgrade 2000.

To explore the possibility of binding aflatoxin B1 to Minazel Plus in IN VITRO conditions To monitor health condition of chickens, clinical symptomatology of intoxication and the timescale of death of chickens To examine whether aflatoxin intoxication has an effect on body mass and weight gain of broilers To examine the pathomorphological and pathohistological changes in organs and tissues of broilers To examine the presence of residues of aflatoxin B1 in organs and tissues of broilers To examine the protective effect of Minazel Plus on poultry exposed to aflatoxin B1

The research work included two experiments: In vitro (laboratory analysis of aflatoxin adsorption) In vivo (broiler farm trial) IN VITRO TRIAL % of aflatoxin binding to Minazel Plus when exposed to different temperature conditions and different ph values. IN VIVO TRIAL Following parameters was monitored: Clinical picture of aflatoxicosis Body weight and weight gain Presence of aflatoxin residues in liver, kidneys, ventriculus, proventriculus, and muscles (dark and light)

Test was carried out in the electrolyte (0.1M HCl and 0.05M NaCl) present in the gastric juice of animals. Electrolyte ph had been adjusted at 2 and 7. Adsorption was examined at 37 C Solution contained 1g of Minazel Plus and the concentrations of aflatoxin B1 50-300 µg. The quantity of unadsorbed mycotoxins had been determined using HPLC method. The adsorption was measured starting from 5 minutes to 48 hours of mycotoxin-adsorbent contact.

Table - Index of aflatoxin B1 adsorption, % Aflatoxin B1 µg/g Contact time 5' 30' 6 h 24 h 48 h ph 2 50 84 84 82 90 90 100 84 83 89 85 87 200 84 82 79 84 86 300 87 78 78 84 87 ph 7 50 86 90 94 96 96 100 87 89 94 94 94 200 84 86 91 93 93 300 87 88 90 93 95

Adsorption index was high (80-95 %) in ph 2 and ph 7, for aflatoxin B1 concentration from 50 to 300 mg/kg. Speed of adsorption was very high, most of aflatoxin was adsorbed during first few minutes. Once adsorbed, aflatoxin B1 has shown no signs od desorption

Trial was done with 50 Hybro broiler chicks Feeding period was 21 days Broiler were fed ad libitum Feed mixture was examined on presence of mycotoxins: aflatoxin, ochratoxin, trichothecenes Broilers were divided in 5 groups (1 control and 4 trial groups) Aflatoxin B1 was applied to broilers in groups (3, 4 and 5) 0.1 mg/kg of life weight, directly, per os.

Group Concentration (mg/kg LW) Aflatoxin B1 mode of application Concentration Minazel Plus mode of application C - - - - T 1 - - 0,3% mixed in feed T 2 0, 1 per/os - - T 3 0,1 per/os 0,3% mixed in feed T 4 0,1 per/os 0,1 g/kg LW per/os

Day of trial Group C T 1 T 2 T 3 T 4 AFB1 - - + + + Minazel Plus - + - + + (per os) 1 808.0 811.2 832.0 789.0 790.0 3 902.2 890.2 944.0 887.0 890.2 5 1034.7 1033.7 1065.8 1018.8 976.8 7 1176.7 1161.8 1197.1 1135.8 1118.4 9 1263.7 1260.7 1283.0 1231.5 1218.3 11 1409.8 1388.7 1403.0 1371.2 1337.4 13 1652.0 1619.5 1568.8 1612.6 1531.1 15 1756.2 1744.2 1680.4 1729.8 1680.2 17 1892.9 1899.8 1754.3 1835.9 1793.9 19 2022.7 2019.9 1863.5 1968.3 1907.5

Day of trial Group C T 1 T 2 T 3 T 4 AFB1 - - + + + Minazel Plus - + - + + (per os) 1-3. 93.7 83.8 112 98.00 100.00 3-5. 137.50 x 138.60 x 121.80 a 131.80 x 84.60 b, y 5-7. 142 120.2 131.3 117 141.6 7-9. 87 98.9 85.9 95.7 99.9 9-11. 146 128 120.9 139.7 118.1 11-13. 238.6 230.8 165.8 241.40 193.7 13-15. 104.2 145.20 x 40 117.2 149.10 x 15-17. 136.7 125.1 73.90 y 106.1 123.7 17-19. 119.80 a 120.1 108.30 b 132.4 113.6 1-19. 1214.20 a 1208.00 a 1037.2 b 1179.3 a 1117.30 b

Group Liver Weight of organ, % Ventriculus C 2,872 1,922 T 1 2,830 1,910 T 2 3,106 1,927 T 3 2,979 1,912 T 4 2,900 1,905

Bird C T 1 T 2 T 3 T 4 - AFB1 - AFB1 + AFB1 + AFB1 + AFB1 - Minazel Plus + Minazel Plus in feed - Minazel Plus + Minazel Plus in feed + Minazel Plus, per os 1 - - + + - + + 2 - - + + - - 3 - - + + - - 4 - - + + - + + 5 - - + + - + + 6 - - + + - - 7 - - + + - - 8 - - + + - - 9 - - + + - - 10 - - + + - - Σ, % 0 0 100 0 30

Bird C T 1 T 2 T 3 T 4 - AFB1 - AFB1 + AFB1 + AFB1 + AFB1 - Minazel Plus + Minazel Plus in feed - Minazel Plus + Minazel Plus in feed + Minazel Plus, per os 1 - - + + - + + 2 - - + + - - 3 - - + + - - 4 - - + + - + + 5 - - + + - + + 6 - - - - - 7 - - + + - - 8 - - - - - 9 - - - - - 10 - - + + - - Σ, % 0 0 70 0 30

Bird C T 1 T 2 T 3 T 4 - AFB1 - AFB1 + AFB1 + AFB1 + AFB1 - Minazel Plus + Minazel Plus in feed - Minazel Plus + Minazel Plus in feed + Minazel Plus, per os 1 - - + + - + + 2 - - + + - - 3 - - + + - - 4 - - + + - + + 5 - - + + - + + 6 - - - - + + 7 - - - - - 8 - - + + - - 9 - - - - - 10 - - + + - - Σ, % 0 0 70 0 40

In birds from the control groups no pathoanatomic or histopathological changes in liver were observed. In birds from the group T-2 (treated with aflatoxin B1) changes in the liver were observed: Bleedings Fatty dystrophy Necrosis In birds from the group T-3 (treated with aflatoxin B1 and Minazel Plus) no pathoanatomic or histopathological changes in liver were observed.

In broilers exposed to Aflatoxin B1 without Minazel Plus: pathoanatomical and pathohistological alterations were present in: Liver Kidneys Spleen Stomach Muscles aflatoxin B1 residues were present in: Liver Kidneys Spleen Stomach Muscles Negative effects on body weight and daily gain were noticed NO ALTERATIONS AND RESIDUES WERE FOUND IN BROILERS EXPOSED TO AFLATOXIN B1 WITH ADDITION ON MINAZEL PLUS! MINAZEL PLUS HAS IMPROVED BODY WEIGHT AND DAILY GAIN PARAMETERS!

EFFECT OF MINAZEL PLUS IN Vera Radović PhD* LAYING HENS * University of Kragujevac, Faculty of Agriculture Čačak2000.

Testing influence of Minazel Plus on: Laying rate Strength of bones Body weight Mortality

Trial was done with 1000 laying hens Isabrown SSL divided into 4 grups: I group C control II group T-I with 0.2% Minazel Plus III group T-II with 0.3% Minazel Plus IV group T-III with 0.5% Minazel Plus Trial duration was 210 days. Trial started in week 46 of age (week 26 of laying period)

GROUP C T-I T-II T-III Number of hens, start 250 250 250 250 Average number of hens 245 246 247 247.50 Total egg production 43 826 43 908 44 299 45 034 Eggs per total hen number 175.30 175.63 177.19 180.14

Group of hens Tibia Average Standard deviation Average Humerus Standard deviation C 20.80 5.02 14.50 3.67 T-I 21.30 3.20 13.40 3.80 T-II 23.70 53.22 13.20 4.19 T-III 20.70 1.67 15.70 0.19

GROUP OF HENS C T-I T-II T-III Total body weight 15,170.00 15,855.00 15,600.00 17,120.00 Average (g) 1,896.25 1,981.87 1,950.00 2,140.00 Index 100.00 104.51 102.83 107.98 Difference +4.51 +2.83 +7.98

Number of hens at the beginning of the experiment GROUP OF HENS C T-I T-II T-III 250 250 250 250 Hen death rate 10 8 6 5 Mortality % 4.0 3.2 2.4 2.0 Index 100 80 60 50 Difference -20% -40% -50%

Trial results show that addition of Minazel Plus in laying hens feed: IMPROVED Laying rate IMPROVED Strength of bones IMPROVED Body weight DECREASED Mortality

EFFECT OF MINAZEL PLUS ON PRODUCTION PARAMETERS IN Dejan R. Karović, dipl. ing * Master Thesis BROILERS * University of Kragujevac, Faculty of Agriculture Čačak2000.

Testing influence of different dosage of Minazel Plus on production parameters of broilers

The examination was done on 300 broilers (COBB 500) divided into 3 groups: K-group (100 broilers without mineral adsorbents ) 0-I group (100 broilers with 0,3% of Minazel Plus) O-II group (100 broilers with 0,5% of Minazel Plus)

Group ( n=100) Age in days K K-control 0-I 0,3% Minazel Plus 0-II 0,5% Minazel Plus Day 1 (average) 39.59 A,B,C 41.20 C 41.18 A Index % 100.00 104.05 104.00 Difference 4.05 4.00 Day 15 546.49 D,E,F 572.90 F 576.39 D,g Index % 100.00 104.05 104.00 Difference 4.05 4.00 Day 30 1284.97 H,I 1322.22 I,K,l 1381.05 H,J,K Index % 100.00 102.90 107.48 Difference 2.90 7.48 Day 42 1950.93 M,n,o 2013.62 o,q,r 2084.44 M,P,Q Index % 100.00 103.21 106.84 Difference 3.21 6.84

Parameter K K-control Group (n=100) O-I 0,3% Minazel Plus O-II 0,5% Minazel Plus Feed consumption, g 3677 3665 3660 Index, % 100.00 99.67 99.54 Difference 0.33 0.46 Feed conversion,g g 2.161 1.891 1.716 Index, % 100.00 87.53 79.41 Difference 12.47 20.59

Trial results show that addition of Minazel Plus in broiler feed has: IMPROVED growth rate IMPROVED body weight DECREASED feed conversion ratio

Efficiency of the surfactant modified clinoptilolite in mitigating the toxic effects of ochratoxin A on performance and meat Dr Olivera Zurovac-Kuzman* PhD Thesis quality of broilers * University of Belgrade, Faculty of Veterinary Medicine Belgrade 2001

1. To provide detailed insight into: production, health condition, slaughter traits, meat quality, presence of residues of ochratoxin in tissue and organs of broilers exposed to ochratoxin A; 2. To determine the possibility of preventing the harmful effects of OTA using Minazel Plus (Patent co.).

To determine effects of OTA to productivity results and health conditions of broilers; To determine influence of OTA on slaughter traits and meat quality of broilers; To detect a hematology and biochemical parameters of treated broilers blood; To determine the presence of residues of OTA in tissues and organs of broilers; To establish involvement in prevention effect of Minazel Plus to OTA contaminated feed of broilers

pure crystal OTA Sigma (O-1877) in concentration of 99% obtained from cultures of Aspergillus ochraceus (303-47-9) for in vitro testing; for the purposes of the experiment in in vivo conditions OTA was extracted from Aspergillus ochraceus NRRL 3174; Minazel-plus- surfactant modified clinoptilolite, (manufacturer Patent co. Belgrade)

The experiment was conducted on 150 one day old broilers, both sexes and Arbor Acres provenience with average body mass weight of 42,48±1.7g; Broiler were divided to 6 groups: I group K I control-without OTA and without Minazel Plus (Mz) II group K-II- 0,5 % Mz added to feed III group O-I- feed with 0,5 mg / kg OTA IV group O-II- feed with with 0,5 mg / kg OTA+ 0,5 % Mz V group- O-III- feed with 1 mg / kg OTA VI group- O-IV- feed with with 1 mg / kg OTA+ 0,5 % Mz

The trial lasted 42 days and experimental groups were treated with chosen substances through whole experiment. During the experiment it was monitored and statistically analyzed : 1. the health condition and production results of broilers; 2. chemical composition of broilers feed; 3. hematological and biochemical blood parameters of broilers; 4. determination of the yield and quality of meat; 5. presence of OTA in meat;

D a y s O f T h e E x p e r i m e n t 1. 14. 28. 42. K-I 42,74±1.83 310,80±47,51 876,40±104,04 1888,56±361,37 K-II 42,50±1,44 313,20±62,9 898,60±88,43 1927,20±276,28 O-! 42,16±1,74 292,80±26,85 845,20±96,27 1831,60±174,04 O-II 42,14±1,86 300,40±44,02 874,00±89,07 1880,00±240,97 O-III 42,50±1,44 276,40±41,12 808,80±87,72 1767,60±135,25 O-IV 42,86±1,88 287,60±33,7 838.80±81,51 1820,40±191,19 Body weight of the group fed with contaminated feed with 0.5 mg / kg OTA in addition Minazel Plus 0.5% was achieved almost equal weight groups fed uncontaminated food without adsorbent

P e r i o d O f T h e E x p e r i m e n t 1-14. 14-28. 28-42 1-42. K-I 1.36 1,93 2,16 1,97 K-II 1,34 1,86 2,12 1,93 O-! 1,52 2,07 2,32 2,13 O-II 1,48 2,00 2,28 2,08 O-III 1,67 2,20 2,44 2,26 O-IV 1,58 2,11 2,37 2,18

Kindey O-I 1,77±0,78 O-II 0,88±0,20 O-III 3,23±0,80 O-IV 1,22±0,42 Liver O-I 1,93±0,53 O-II 1,17±0,37 O-III 2,88±0,93 O-IV 1,73±0,16

Breasts O-I 0,40±0,09 O-II 0,30±0,06 O-III 0,72±0,26 O-IV 0,70±0,09 Drumsticks O-I 0,65±0,14 O-II 0,50±0,09 O-III 1,03±0,76 O-IV 0,57±0,19

1. Minazel Plus decrease symptoms of mycotoxicosis occurred with use of feed contaminated with concentration of ochratoxin A 0,5 and 1,0 ppm (sporadic diarrhea and increased intake of water) 2. Lower production results (growth lower for 6.11 to 14.77%, lower consumption of 4,52-12.56% conversion lower for 0.51 to 9.14%), can be improved by Minazel Plus.

4. The use of Minazel Plus normalize in certain extant previously altered biochemical and blood parameters during fed of OTA contaminated feed; 5. By adding a Minazel Plus to contaminated mixtures for a shorter consumption period, the negative effects on the development of alterations in liver were effectively mitigated. 6. Quantity of residues of OTA in the liver, kidneys, thighs and breasts is proportional to the content of toxins in animal feed and presence of residues is significantly reduced by using of Minazel Plus.

Negative effects of ochratoxin A on the performance and health of broilers can be mitigated with use of Minazel Plus in feed for broilers.

Effectiveness of different adsorbents added to feed in alleviating harmful effects of Ochratoxin A in broilers Dr Jelena Nedeljković-Trailović * PhD Thesis * University of Belgrade, Faculty of Veterinary Medicine Belgrade 2007

Ochratoxin A (OTA) is a toxic product of fungi from the genus Aspergillus and Penicillium. It was isolated from almost all grain nutrients, but also from the complete mixtures for animal nutrition. (Balzer et al. 1978) OTA presence in feed and animal tissue means that circling through the food chain will inevitably endanger humans. (Trailovic, 2007) Conditions: OH O COOH Production 7-14 days Moisture > 16% CH 2 CH NH CO O H Air humidity > 70% CH Temperature: 20-25 C 3 Cl H Warehouse mould H

1. Acute ochratoxicosis - infrequent Listlessness, huddling, occasionally diarrhea and prostration; (Prior et al., 1976) the most sensitive indicator of acute ochratoxicosis poultry is alterations in levels of total protein and albumin; 2. Chronic ochratoxicosis - frequent poor condition, ataxia, abdominal enlargement, depression, diarrhea, slow growth and increase in the percentage of mortality; ( Slonserberg et al., 1997)

Decreased egg production, thin or soft egg shells and poor growth rate; (Hamilton et al. 1982) Significantly lower body weight, lower average daily gain and higher feed conversion ratio; (Harvey et al., 1987., Trailovic et al. 2004, Acta veterinaria)

1. To provide detailed insight into: the performance; health status; histopathological changes in tissues and organs of broilers exposed to OTA; 2. To determine the possibility of preventing the harmful effects of OTA using Minazel Plus.

1. To test adsorption or deactivation of OTA within Minazel Plus in in vitro conditions: when an intestinal juice of broilers is medium; by everted duodenum method. 2. To test in in vivo conditions the level of protective effects of Minazel Plus during alleviation of the adverse effects of OTA, in feed for broilers on their health status and pathological changes in the target organs;

1. Standard OTA (Biopur, Austria) with purity of 99%. 2. Surfactant modified clinoptilolite- (Minazel Plus, manufacturer Patent co. Belgrade), (Mz)

Tubes containing toxin (2μg/ml) and 100mg of each adsorbent ( were placed for 30 min in a 37 C water bath equipped with a horizontal shaker, shaking frequency 50-60 cycles per minute. After 30min, samples were centrifuged for 10 min and 2 ml of the aqueous supernatant was used for HPLC analysis. Percentage of adsorption, i.e. adsorbent efficiency, was in the presence or absence of adsorbent determined by comparing initial concentration of OTA, with concentration of toxin measured.

Investigations were carried out on duodenal segments of 24 broilers (Ross proveniencee) BW 850 to 1000 g; Duodelan segments 7 to 9 cm long were immediately before incubation inverted by mean of glass rod, so that absorptive surphase (mucose) has left outside; Intestines were kept in oxigenated 95%O2/5%CO2 Mes-Krebsov solution (mm): 15.5 Mes, 120.7 NaCl, 5.6 KCl, 2.5 CaCl2, 1.2 MgCl2 i 11.5 glucose, ph 6.0.

One side of intestinene was carefully ligated by surgical suture and lumen was filled with 5 ml of Mes-Krebsov solution (serosal compartment). The other end of intestine was then closed with another surgical suture; Glasses with samples were incubated for 1 hour in a water bath with a movable bottom at a temperature of 39-40oC and bottom of the frequency shift (mixing) of 50-60 per minute;

Six inverted gut segments were placed in glass beakers with 20 ml of media /Mes-Krebsov solution/ (Mucosal compartment) which was spiked with OTA to a final concentration of 2 g/ml.

Beakers with everted intestinal segments were incubated for 1 h in water bath (HAAKE SWB 20) T 39 C and shaker 50 RPM Tissue was at all times oxigenated by 95%O 2 /5%CO 2

The experiment was conducted on 120 one day old broilers, both sexes and Ross provenience; Broiler were divided to 4 groups: I group C control II group E-I- feed with 2 mg / kg OTA III group E-II- feed with 2 mg / kg OTA + 0.2% surfactant modified cliniptilolite adsorbent (Mz) IV group- E-III feed with surfactant modified cliniptilolite adsorbent (Mz)

The trial lasted 42 days and experimental groups were treated with chosen substances to 21th day of the experiment and after that by the end of the experiment were fed free of toxin and adsorbent.

OA in the intestinal juice 2 mg / ml Control, intestinal juice without adsorbent Surfactant modified cliniptilolite(mz) Concentration of toxins in the intestinal juice after one hour incubation with adsorbent (mg / ml) 1.94 0.05 0.26 0.03 ooo (P<0.0001) % of adsorbed mycotoxin 0% 80.86 1.85

Mz Absorption % % of adsorption OTA ( g/ml) 100 75 50 25 0 80.86% 2.5 2.0 1.5 1.0 0.5 0.0 Intestinal fluid 1.94 g/ml 0.26 g/ml Mz ooo Trailović results on the efficiency of surfactant modified clinoptilolite are consistent to Daković et al. (2001) study, where surfactant modified clinoptilolit at ph 7 in vitro adsorbed 87% of OTA Mz- surfactant modified clinoptilolite adsorbent (Mz)

OTA Standard 1.2 ng OTA concentration in intestinal fluid after incubation with Minazel Plus

OTA 2 mg/ml Control Surfactant modified clinoptilolite adsorbent (Mz ) Concentrations of absorbed toxins in serous compartment of duodenum (mg/ml) 0.0299 0.0044 0.0157 0.0020 * *-P=0.0156 % of adsobtion 0 45.75 7.14 * -statistically significant difference compared to the control test Minazel Plus significantly reduced absorption of OTA from the medium into the intestine

OTA ( g/ml) 0.04 0.03 0.02 0.01 0.00 0.029 g/ml OTA * 0.015 g/ml 45.75% Mz According to Trailovic references, there is no available literature related to overturned duodenum broiler type of testing for possible comparison with the results obtained in her study Mz- Minazel Plus

Pale and enlarged kidneys with heamorhage E-I group fed with 2mg/kg OTA

Diffuse accumulation of fat in hepatocytes Focal accumulation of fat in hepatocytes Group fed with 2mg/kg OTA Group fed with 2mg/kg OTA +Minazel Plus

Necrotic areas in tubulocites Edema of the proximal canaliculi kidney cells with vacuolization and partial lumen reduction of tubulocites Group fed with 2mg/kg OTA Group fed with 2mg/kg OTA +Minazel Plus

Restitution changes in the liver Regenerative changes in of tubulocytes with the appearance of mitosis in cells and glomerual sclerotic changes in the form of capsules and thickening of the vascular loop Both groups Group fed with 2mg/kg OTA +Minazel Plus

1. In in vitro studies in intestinal juice the extreme efficiency in the adsorption of ochratoxin A (OTA) is exhibited by Minazel Plus (80.86% adsorption) ; 2. Minazel Plus significantly reduced absorption of OTA from the medium into the intestine. 3. Minazel Plus was added to the feed at the recommended concentration of broiler producers for three weeks, showed no negative effects on the health of the treated animals.

4. Adding Minazel Plus in broiler feed that contained OTA reduces the incidence of mortality and morbidity as well as the intensity of the clinical symptoms of the disease, compared to broilers that received food only OTA. 5. Pathohistological changes observed liver and kidneys in this study were most prominent in group of broilers fed 2 mg/kg of OTA while these changes were of much lower intensity in the group supplemented with Minazel Plus.