Haematotoxicity of dietary Fumonisin B1 in growing pigs
Investigation was carried out with twenty-four (24) crossbred (Large White x Landrace) male weanling pigs of 8-9 weeks of age averaging 6.94±0.26 kg to study the haematotoxicity of dietary fumonisin B1 (FB1) in pigs. The 24 animals were randomly assigned to four treatment diets containing approximately 0.10, 5.10, 10.10 and 15.10 ppm fumonisin B1 constituting diets 1,2,3 and 4 respectively, in a 6-month feeding trial. Blood sample was collected from the ear vein of each animal at the end of the feeding trial for haematological analysis. Dietary FB1 has significant influence on haematological parameters. Animals fed diets 3 and 4 suffered significantly (P<0.05) reduced erythrogenesis and concentration of red blood cells, and consequent anaemia, with impaired respiratory capacity. The animals exposed to diet 4 (containing the highest FB1 concentration) suffered leukocytosis. Dietary FB1 concentration above 5.0ppm is haematotoxic in pigs. The study suggests that the USFDA recommended a maximum level of 10 ppm total fumonisins is above the no observable effect level (NOEL) for swine.
Key words: Erythrogenesis, fumonisin B1, haematotoxicity, leukocytosis, pig.
Running Title: Effect of fumonisin B1 on haematology.1. INTRODUCTION
The contamination of feeds and feedstuffs with mutagenic and carcinogenic mycotoxins is a major concern for animal and human health. Mycotoxins are formed where environmental factors are conducive, during the growth of frequently occurring mycomycetes on foodstuffs and animal feeds, the process taking place during the secondary metabolism.
Fumonisins are a group of environmental mycotoxins produced by various species of Fusarium, especially Fusarium verticillioides (= F. moniliforme), one of the most prevalent seed-borne fungi associated with maize intended for human and animal consumption throughout the world (Nelson et al., 1991; Kedera et al., 1992). The major types of fumonisins are B1, B2 and B3, and have been implicated in modification of immune response. Shephard et al. (1996) reported that maize, which is the major cereal utilized in the formulation of livestock feeds as well as a major dietary staple in several parts of the world, is the only commodity that contains significant amounts of fumonisins; hence the potential for fumonisins to be found in feeds and foodstuffs is high. A survey of contemporary literature reveals increasing wave of fumonisin contamination of feeds and feeding stuffs (Fazekas et al., 1997) and consequent poisoning of a large number of animals in several parts of the world.
In general, the consumption of mycotoxin-contaminated feed by an animal may result in an unhealthy situation ranging from decreased nutritive value of feeds, poor feed conversion, reduced growth, hormonal changes to occasional organ damage or even cause death depending on the type of mycotoxin. The symptoms depend on the amount of toxin in the feed, the period for which the feed is ingested, the nutritional status of the feed and the consumption of sufficient quantities of toxins–containing plant material (Marasas and Nelson, 1987).
The blood profile has been shown (Wilson and Medd, 1978) to be a guide to nutritional status in feeds. The blood contains a myriad of metabolites and other constituents, which provide a valuable medium for clinical investigation and assessment of nutritional status of human beings and animals. Hence, the use of blood and serum biochemical parameters in medical nutritional assessment and survey of animals (Olorode et al., 1995). Fumonisin B1 (FB1) is the major fumonisin produced in culture (Bezuidenhout et al., 1988; Ross et al., 1992) as well as naturally occurring in maize and maize-based feeds and foods (Rheeder et al., 1992; Ross et al., 1992). Consequently, toxicological studies on the fumonisins have concentrated on FB1. With these in mind, coupled with the fact that fumonisins have been reported to cause different physiological responses in animals, this study was designed to assess the effect of varied dietary FB1 on haematological parameters in growing pig.
2. MATERIALS AND METHODS
Fumonisin–contaminated maize grains, cultured with Fusarium verticillioides, was generated according to the method described by Nelson and Ross (1992) at the Plant Pathology Laboratory, International Institute of Tropical Agriculture (IITA), Ibadan, Nigeria. Three treatment diets containing approximately 5.10, 10.10 and 15.10ppm FB1 constituting diets 2, 3, and 4 respectively were formulated. With diet 1 (control), the treatment diets were used in a six-month feeding trial. The varied dietary FB1 concentrations were achieved by substituting ground cultured maize for ground, autoclaved noncultured maize in various proportions. The FB1 concentrations were determined using the fumonisin qualitative test kit (Neorgen Corp., USA)
Twenty four Large White x Landrace male weanling pigs (about 8 - 9 weeks of age) were randomly, in a completely randomized design, assigned to each of the 4 treatment diets, such that each treatment had 6 animals. The feeding trial was divided into 3 physiological phases (weanling, peri-pubertal and pubertal). The gross composition of the treatment diets fed during weanling, grower and finisher phases (for 6, 10 and 8 weeks respectively) are shown in Tables 1 - 3.
The animals were fed their respective diets ad libitum daily at 0800h and 1600h. Potable water was made available throughout the experimental period. At the end of the feeding trial, blood sample was collected from the ear vein of each animal into Monoject® vacutainer containing Ethylene diaminetetraacetic acid (EDTA) for haematological analysis.
A portion of each blood sample was centrifuged in a capillary tube for 5 minutes in a haematocrit centrifuge and read in a haematocrit reader to determine the packed cell volume (PCV) while the erythrocyte , leukocyte and the differential leukocyte counts were determined using the new improved Neubauer haemocytometer. The haemoglobin concentration and the blood constants: mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC) were determined using cyanethaemoglobin method and appropriate formula respectively as described by Jain (1986).
Data obtained were subjected to one way ANOVA procedure of SAS® (1999) and the significant treatment means separated by Duncan option of the same software.
3. RESULTS AND DISCUSSION
The haematological indices of pubertal boars exposed to varied levels of dietary FB1 for 24 weeks are shown in Table 4. All the parameters monitored, except the blood constants and the platelets, were significantly (P<0.05) influenced, with the erythrocytes, haemoglobin, PCV and the leukocytes revealing a dose-dependent significant variations across the treatments. The erythrocytes, haemoglobin, MCV, MCH, and the PCV values generally decreased with increase in the dietary FB1. The platelets and the leukocyte values, on the other hand, increased while all other parameters did not follow any particular trend with increases in the dietary FB1 concentrations.
Blood is an important index of physiological and pathological changes in an organism, and it is also used in assessing the body’s ability to respond to haematological insult. The results revealed that the animals exposed to feeds containing Fusarium–inoculated maize (diets 2, 3 and 4) suffered significantly from the synthesis (erythrogenesis) and concentration of red blood cells (RBCs). The corresponding statistical decrease in PCV of the animals exposed to diets 2, 3 and 4 revealed that the animals were anaemic. The significantly reduced PCV values were directly related to the FB1 concentration in the diets, with the animals that consumed diet containing the highest dietary FB1 concentration recording the lowest value.
The values of the haematological indices- MCV and MCH, were within the reported reference ranges of 50 – 68 fl and 17.8 – 18.8 µµg by Thorn (2000) and Mitruka and Rawnsley (1977) respectively for young adult boars. The characteristic normal MCV and MCH with decreased number of erythrocytes and low PCV observed for animals fed diets 2, 3 and 4 indicated normocytic anaemia.
Haemoglobin (Hb), an iron-containing conjugated protein has been described (Mitruka and Rawnsley, 1977) to have physiological function of transporting oxygen and carbon dioxide. Although the significantly lower Hb values of 112.60 and 122.90 g/L recorded for animals on diets 3 and 4 compared with those on diets 1 and 2 are within the 100 – 160 g/L reference values for young adult boars reported by Blood (1995) and Thorn (2000), the results suggested that the animals fed diets 3 and 4 suffered depressed respiratory capability. This further confirms the low respiratory and gas-exchange capacity of the RBCs in animals exposed to diets 3 and 4, which have been suggested earlier in this report to have suffered from normocytic anaemia.
The non-significant difference observed for the platelets across the treatments revealed that the consumption of the dietary fumonisin, the concentration not withstanding, did not cause splenic atrophy– a characteristic pathologic thrombocytosis, neither did the animals suffered bone marrow damage nor marrow failure which, among other factors, has been reported (Coles, 1986) to cause thrombocytopenia.
The circulating leukocyte count, which was significantly higher in animals fed diet 4 than in those fed diets 1,2 and 3, was higher than the 11.0 – 22.0x109/L reference values (Blood, 1995; Thorn, 2000) in animals exposed to diet containing the highest FB1 concentration. This indicates that the animals fed diet 4, containing the highest concentration of FB1, suffered from leukocytosis, which Coles (1986) described as a consequence of an increase in the total number of circulating neutrophils, which is highest for the animals on diet 4. Leukocytosis, according to Coles (1986), may result from intoxications including those produced by metabolic disturbances among others.
The generally significant influence of the dietary treatment on most of the haematological parameters investigated agrees with the findings of Rotter et al. (1996) that reported changes in selected haematological parameters in pigs at dietary levels as low as 1mg/kg pure fumonisin and those of Espada et al. (1997) who revealed altered hematological parameters in broiler diets formulated from F. verticillioides culture material containing 10mg/kg and 30mg/kg fumonisin B1. These results, however, contradict the report of Parent–Masin and Parchment (1998), Zomborszky–Kovács et al. (2002) and Ogunlade et al. (2004) that fumonisins are considered as non–haematotoxic mycotoxins. The variability in haematology of livestock fed fumonisin contaminated diets might be the result of different doses of fumonisin, the species of livestock used, or the length of exposure of such livestock to the mycotoxin.
The dietary FB1 concentrations used in this study bracketed the published FDA recommended maximum level of 10ppm total fumonisins for swine (USFDA, 2001). This study has shown that dietary exposure to FB1 at a concentration of about 10ppm or more for a six-month period may result in significantly reduced synthesis and concentration of RBC and consequent anaemia with impaired respiratory capacity in boars. The results in this study suggest that the FDA recommended maximum levels of 10ppm are above the no observable effect level (NOEL) for swine. The study also demonstrates that boars exposed to dietary fumonisin concentration of about 15ppm will suffer from leukocytosis.
Table 1: Gross composition (%) of the weanling pigs’ test diets
| Diet 1 | Diet 2 | Diet 3 | Diet 4 | |
| Ingredient | 0.10ppm | 5.09ppm | 10.08ppm | 15.07ppm |
| Non-inoculated maize | 40.00 | 38.26 | 36.52 | 34.78 |
| Inoculated maize* | - | 1.74 | 3.48 | 5.22 |
| Soybean meal | 20.00 | 20.00 | 20.00 | 20.00 |
| Palm kernel cake | 20.00 | 20.00 | 20.00 | 20.00 |
| Wheat offal | 14.30 | 14.30 | 14.30 | 14.30 |
| Fish meal | 3.00 | 3.00 | 3.00 | 3.00 |
| Dicalcium Phosphate | 1.50 | 1.50 | 1.50 | 1.50 |
| Oyster shell | 0.50 | 0.50 | 0.50 | 0.50 |
| Salt | 0.45 | 0.45 | 0.45 | 0.45 |
| Premix (Starter’s) | 0.20 | 0.20 | 0.20 | 0.20 |
| Methionine | 0.01 | 0.01 | 0.01 | 0.01 |
| Lysine | 0.04 | 0.04 | 0.04 | 0.04 |
| Total | 100.00 | 100.00 | 100.00 | 100.00 |
| Calculated nutrients: | ||||
| Crude Fibre (%) | 5.35 | 5.35 | 5.35 | 5.35 |
| Crude Protein (%) | 20.38 | 20.38 | 20.38 | 20.38 |
| DE**(Kcal/Kg) | 2701.80 | 2701.80 | 2701.80 | 2701.80 |
*Inoculated with Fusarium verticillioides; **Digestible Energy.
Table 2: Gross composition (%) of the peri-pubertal pig test diets
| Diet 1 | Diet 2 | Diet 3 | Diet 4 | |
| Ingredient | 0.10ppm | 5.09ppm | 10.08ppm | 15.07ppm |
| Non-inoculated maize | 30.00 | 28.26 | 26.52 | 24.78 |
| Inoculated maize* | - | 1.74 | 3.48 | 5.22 |
| Palm kernel cake | 25.00 | 25.00 | 25.00 | 25.00 |
| Soybean meal | 15.00 | 15.00 | 15.00 | 15.00 |
| Wheat offal | 14.30 | 14.30 | 14.30 | 14.30 |
| Rice husk | 11.00 | 11.00 | 11.00 | 11.00 |
| Fish meal | 2.00 | 2.00 | 2.00 | 2.00 |
| Dicalcium Phosphate | 1.50 | 1.50 | 1.50 | 1.50 |
| Oyster shell | 0.50 | 0.50 | 0.50 | 0.50 |
| Salt | 0.45 | 0.45 | 0.45 | 0.45 |
| Premix (Grower’s) | 0.20 | 0.20 | 0.20 | 0.20 |
| Methionine | 0.01 | 0.01 | 0.01 | 0.01 |
| Lysine | 0.04 | 0.04 | 0.04 | 0.04 |
| Total | 100.00 | 100.00 | 100.00 | 100.00 |
| Calculated nutrients: | ||||
| Crude Fibre (%) | 9.82 | 9.82 | 9.82 | 9.82 |
| Crude protein (%) | 17.97 | 17.97 | 17.97 | 17.97 |
| DE**(Kcal/Kg) | 2269.11 | 2269.11 | 2269.11 | 2269.11 |
*Inoculated with Fusarium verticillioides; **Digestible Energy.
Table 3: Gross composition (%) of the pubertal pig test diets
| Diet 1 | Diet 2 | Diet 3 | Diet 4 | |
| Ingredient | 0.10ppm | 5.09ppm | 10.08ppm | 15.07ppm |
| Non-inoculated maize | 20.00 | 18.26 | 16.52 | 14.78 |
| Inoculated maize* | - | 1.74 | 3.48 | 5.22 |
| Palm kernel cake | 45.00 | 45.00 | 45.00 | 45.00 |
| Rice husk | 17.80 | 17.80 | 17.80 | 17.80 |
| Soybean meal | 8.50 | 8.50 | 8.50 | 8.50 |
| Wheat offal | 5.00 | 5.00 | 5.00 | 5.00 |
| Fish meal | 1.00 | 1.00 | 1.00 | 1.00 |
| Dicalcium Phosphate | 1.50 | 1.50 | 1.50 | 1.50 |
| Oyster shell | 0.50 | 0.50 | 0.50 | 0.50 |
| Salt | 0.45 | 0.45 | 0.45 | 0.45 |
| Premix | 0.20 | 0.20 | 0.20 | 0.20 |
| Methionine | 0.01 | 0.01 | 0.01 | 0.01 |
| Lysine | 0.04 | 0.04 | 0.04 | 0.04 |
| Total | 100.00 | 100.00 | 100.00 | 100.00 |
| Calculated nutrients: | ||||
| Crude Fibre (%) | 10.83 | 10.83 | 10.83 | 10.83 |
| Crude Protein (%) | 15.30 | 15.30 | 15.30 | 15.30 |
| DE**(Kcal/Kg) | 2240.61 | 2240.61 | 2240.61 | 2240.61 |
*Inoculated with Fusarium verticillioides; **Digestible Energy.
Table 4: Haematolgical indices of pubertal boars exposed to varied levels of dietary FB1.
| Dietary fumonisin level | |||||
| Parameter | 1(0.10ppm) | 2(5.10ppm) | 3(10.10ppm) | 4(15.10ppm) | SEm |
| Erythrocytes (1012/L) | 7.42a | 7.28a | 6.13b | 6.68ab | 0.13 |
| Haemoglobin (g/L) | 136.10a | 133.60a | 112.60b | 122.90ab | 2.30 |
| Mean Cell Volume (fl) | 50.12 | 50.48 | 57.43 | 51.53 | 1.19 |
| MCH * (µµ/g) | 18.35 | 18.35 | 18.36 | 18.40 | 0.01 |
| MCHC** (%) | 36.96 | 37.13 | 32.89 | 36.52 | 0.72 |
| PCV*** (%) | 36.83a | 36.00ab | 34.33b | 33.83b | 0.32 |
| Platelets (109/L) | 20.03 | 21.68 | 20.42 | 20.60 | 0.38 |
| Leukocytes (109/L) | 15.28b | 15.42b | 18.37b | 22.52a | 0.52 |
| Neutrophils (109/L) | 2.01ab | 2.91a | 1.87b | 2.99a | 0.24 |
| Eosinophils (109/L) | 0.37ab | 0.46ab | 0.27b | 0.69a | 0.03 |
| Lymphocytes (109/L) | 12.69b | 11.81b | 16.34a | 18.41a | 3.00 |
| Monocytes (109/L) | 0.21b | 0.23ab | 0.09b | 0.44a | 0.02 |
ab Means in the same row with different superscripts differ significantly (P<0.05).
*Mean Corpuscular Haemoglobin; **Mean Corpuscular Haemoglobin Concentration; ***Packed Cell Volume.
1Department of Environmental Biology & Fisheries, AdekunleAjasinUniversity, Akungba – Akoko, Nigeria.
2Animal Physiology Laboratory, Department of Animal Science, University of Ibadan, Ibadan, Nigeria.
The research work was conducted at the Swine Breeding Section of the Animal Physiology Unit of the University Teaching and Research Farm, University of Ibadan. The mycotoxin used for the project was cultured in association with the team of Pathologists at the International Institute for Tropical Agriculture (IITA) Ibadan, Nigeria.
Bezuidenhout S C, Gelderblom W C A, Gorst-Allman C P, Horak R M, Marasas W F O, Spiteller G and Vleggaar R 1988 Structure elucidation of the fumonisins, mycotoxins from Fusarium moniliforme; J. Chem. Soc. Chem. Commun. 1988 743–745
Blood D C 1995 Pocket Companion to Veterinary Medicine (Philadelphia, USA: Bailliere Tindall)
Coles E H 1986 Veterinary Clinical Pathology, 4th Ed (Philadelphia, USA: W. B. Saunders Company)
Espada Y, Ruiz de Gopegui R, Cuadradas C and Cabañes F J 1997 Fumonisin mycotoxicoses on broilers: plasma protein and coagulation modifications; Avian Dis. 41 23-79
Fazekas B, Bajmócy E, Glávits R and Fenyvesi A 1997 Fumonisin mycotoxicoses in Hungary. Leucoencephalomalacia in horses, fattening pulmonary oederma in pigs (in Hungarian); Magy. Allatorv. Lapja. 199 137–139
Jain N C 1986 Schalms Veterinary Haematology, 4th Ed (Philadelphia, USA: Lea and Febiger)
Kedera C J, Leslie J F and Claflin L E 1992 Systemic infection of corn by F. moniliforme. (Abstr.) Phytopathology, 82 1138
Marasas W F O and Nelson P E 1987 Mycotoxicology: Introduction to the Mycology, Plant Pathology, Chemistry, Toxicology and Pathology of Naturally occurring Mycotoxicoses in Animals and Man (University Park: Penn. State Univ. Press) 102 pp
Mitruka B M and Rawnsley H M 1977 Clinical Biochemical and Haematological Reference Values in Normal Experimental Animals (New York: Masson Pub. USA Inc.) pp 104-106
Nelson P E, Plattner R D, Shackelford D D and Desjardins A E 1991 Production of fumonisins by Fusarium moniliforme strains from various substrates and geographic areas; Appl. Environ. Microbiol. 57 2410-2412
Nelson P E and Ross P F 1992 Fumonisin production by Fusarium species on solid substrates; Abstr. 104, 106th AOAC Ann. Meet., Cincinnati, OH., Aug. 31- Sept. 2
Ogunlade J T, Gbore F A, Ewuola E O, Idahor K O, Salako A O and Egbunike G N 2004 Biochemical and haematological responses of rabbits fed diets containing micro doses of fumonisin; Trop. J. Anim. Sci. 7 (1) 169-176
Olorode B R, Ajagbonna O P and Babatunde G M 1995 Comparism of air dried poultry droppings in broiler rations. Effects on performance, organ weight and haematological parameters; Int. J. Anim. Sci. 10 289-293
Parent-Masin D and Parchment R E 1998 Haematotoxicity of mycotoxins; in Mycotoxins in the food chain (eds) J Le Bass and P Galtier (MYCOTOX’98, Toulouse, July2-4) pp 591-598
Rheeder J P, Marasas W F O, Thiel P G, Sydenham E W, Shephard G S and Van Schalkwyk D J 1992 Fusarium moniliforme and fumonisins in corn in relation to human esophageal cancer in Transkei; Phytopathology, 82 353–357
Ross P F, Rice L G, Osweiler G D, Nelson P E, Richard J L and Wilson T M 1992 A review and update of animal toxicoses associated with fumonisin-contaminated feeds and production of fumonisins by Fusarium isolates; Mycopathology, 117 109–114
Rotter B A, Thompson B K, Prelusky D B, Trenholm H L, Stewart B, Miller J D and Savard M E 1996 Response of growing swine to dietary exposure to pure fumonisin B1 during an eight-week period: Growth and clinical parameters. Nat. Toxins. 4 326-329
SAS Institute Inc. 1999 SAS/STAT User’s Guide. Version 8 for windows (SAS Campus Drive, Cary, North Carolina, USA: SAS Institute Inc.)
Shephard G S, Thiel P G, Stockenström S and Syndenham E W 1996 Worldwide survey of fumonisin contamination of corn and corn-based products; J. Assoc. Off. Anal. Chem. Int. 79 671-687
Thorn C E 2000 Normal hematology of the pig; in Schalm’s Veterinary Hematology, 5th ed. (eds) B F Feldman, J G Zinkl and N C Jain (Philedelphia: Lippincott Williams & Wilkins, A Wolters Kluwer Company) pp 1089–1095
U.S. Food and Drug Administration 2001 Center for Food Safety and Applied Nutrition, and Center for Veterinary Medicine, Guidance for Industry. Fumonisin Levels in Human Foods and Animal Feeds. November 9.
http://www.cfsan.fda.gov/~dms/fumongu2.html
Wilson P N and Medds R K 1978 Blood profile as a guide to nutritional status in animals; Brit. Soc. Ani. Prod. 1 155-162
Zomborszky-Kovács M, Kovács F, Horn P, Vetési F, Repa I, Tornyos G and Tóth Á 2002 Investigations into the time– and dose-dependent effect of fumonisin B1 in order to determine tolerable limit values in pigs. Livestock Prod. Sci. 76 251-256
