Abstract: The aim of present study was to determine the species incidence of genus Fusarium in animal and poultry feed mixtures and to know the extent of potential risk of feed contamination by Fusarium mycotoxins. One hundred and seven different animal feed samples and (71) poultry-feed mixtures were collected from Karnataka (India) during April 2004 to April 2005. All samples were analyzed for the incidence of Fusarium species on PDA, DCPA and MGA 2.5 media. A total frequency of the Fusarium species isolated was determined to be 50% and their counts ranged from 9.5x101 to 4.4x105 CFU g-1 of poultry feed and 5.728x101 to 2.088x105 CFU g-1 of animal feed sample. Of the total number of Fusarium isolates (330) from animal and poultry feedstuffs, F. verticillioides recorded 89.09%, followed by F. pallidoroseum (6.66%), F. oxysporum (3.63%) and F. solani (0.6%). The results of this study showed a high incidence of F. verticillioides in poultry feed mixtures while animal feeds especially cotton seeds, fine wheat bran and maize pellets showed high incidence of F. verticillioides. Bengal gram husk, coarse horse gram powder, groundnut seed cake, sunflower seed cake and wheat flakes showed very low incidence of F. verticillioides. The study not only reveals a high incidence of the potentially toxigenic F. verticillioides, in the local feeds of Karnataka region but also represents the possibility of occurrence of fusarial mycotoxins, especially fumonisins.
INTRODUCTION
There are thousands of poultry farms and local animal feed manufacturing units in India, producing mixed feeds for poultry and animal nutrition. In general, the mixed feeds constitute maize and sorghum as major ingredients, which represent an excellent substrate for growth and reproduction of numerous fungi, under favourable conditions such as high moisture and increased temperature (Labuda et al., 2003). A number of morphologically related Fusarium species namely: Fusarium verticillioides (Syn. F. moniliforme), F. proliferatum, F. napiforme, F. anthophilum, F. dlamini, F. thapsinum and F. globosum occur world-wide and are capable of producing a group of structurally related mycotoxins such as fumonisins (Gelderblom et al., 1988). Fumonisins are a group of secondary metabolites produced by F. verticillioides (Nelson et al., 1991). Fumonisin B1 is the most common, most toxic compound occurring on wheat seedlings (Chelkowski et al., 1995) and mainly on maize and maize products (Gutema et al., 2000). Fumonisin containing culture material of F. verticillioides was found to be hepatotoxic and carcinogenic (Kellerman et al., 1990) and nephrotoxic in rats (Suzuki et al., 1995). Fumonisin B1 causes leucoencephalomalacia in horses (Kellerman et al., 1990), pulmonary oedema and hydrothorax in swine (Harrison et al., 1990). Acute hepatic and renal toxicity with significant mortality was found in lambs dosed with fumonisin B1 (Gelderblom et al., 1991). Fumonisins are toxic to fish where enteritis was observed in catfish (Ictalurus punctatus) fed with fumonisin B1 (Lumlertdacha et al., 1995). Feeding F. verticillioides culture material to broilers led to reduced performance, increased organ weights, hepatic necrosis, black sticky diarrhoea, rickets and altered serum constituents and enzyme activities (Weibking et al., 1993). Acute mycotoxicosis caused by the fumonisins or their toxic effects in poultry were studied for instance by other scientific reports (Prathapkumar et al., 1997; Weibking et al., 1993). In spite of the worldwide natural occurrence of fumonisins in maize (Zea mays), which is a major dietary ingredient in poultry feeds, no natural disease outbreak in poultry attributable to fumonisin was reported in India till 1994. However, during October 1995, a poultry disease outbreak was reported from two layer farms more than 100 miles apart (Prathapkumar et al., 1997). Not much work has been done on high-energetic cereals such as maize, sorghum, wheat, which form the bulk ingredients in the production of animal and poultry feed mixtures in Karnataka. Hence the aim of this present study was therefore to determine the species incidence of the genus Fusarium in animal and poultry feed mixtures produced in the region of Karnataka, India and to know the extent of potential risk of feed contamination by Fusarium mycotoxins, especially fumonisins.
MATERIALS AND METHODS
Sample collection: A field trip was undertaken, in order to collect 71 samples of local poultry feed mixtures from various poultry farms in and around Mysore district and also from different districts of Karnataka State during the period of April 2004 to April 2005. One hundred and seven animal feed samples were also collected from local merchants and brought to the laboratory in sterile polythene bags during the same period. Representative samples were obtained by hand sampling for further analysis.
Mycological analysis of poultry and animal feeds: All the samples were subjected to serial dilution and plating. Each sample (1 g) was added to 9 mL of sterile saline and vortexed for 10 min. One mL suspension was spread onto an agar plate in dilution of 10-2 to 10-5 in duplicates. For isolation and enumeration of Fusarium species, Dichloran Chloramphenicol Peptone Agar (DCPA) was used (Leslie and Summerell, 2006). Plates were incubated at 25±2°C, for a period of 6 days with alternating periods of 12 h light and 12 h darkness. The colonies of Fusarium were counted and expressed as Colony Forming Units (CFU) g-1 of the sample. Malachite Green Agar 2.5 (MGA 2.5) medium was also used for selective isolation of Fusarium species (Bragulat et al., 2004). Cottonseeds from 100 g samples were surface sterilized with 1% NaOCl for 30 sec and then plated on Malachite Green agar (MGA 2.5) media. Percent incidence of Fusarium species was calculated using the formula.
Fusarium isolates were sub-cultured and incubated at 25°C, for 10 days in alternating periods of 12 h darkness and light using a carbohydrate-rich medium like Potato Dextrose Agar (PDA) that contains 20 g of dextrose, 20 g of agar and the broth from 250 g white potatoes made up to 1 L with water, supplemented with antibiotics to inhibit bacterial growth. The potatoes were peeled, washed, diced and boiled until soft. The boiled potatoes were filtered through a single layer of cheesecloth and the broth was used. Individual species were identified on the basis of their macro morphological and micro morphological characters in accordance with previously described Fusarium keys (Booth et al., 1977; Leslie and Summerell, 2006).
RESULTS AND DISCUSSION
Out of 107 animal feed samples that were screened, 35 samples were found to be positive (Table 1) and 54 poultry feed mixture samples out of 71 samples tested positive for Fusarium species contamination (Table 2). Their counts ranged from 7.33x102 to 4.58x105 CFU g-1 of animal feed sample. Out of the 175 isolates of the genus Fusarium, four Fusarium species were recorded. F. verticillioides was the most frequently isolated species among the 34 positive samples (Table 3).
On the other hand the total counts of Fusarium species from poultry feed mixture samples ranged from 4.04x102 to 1.95x105 CFU g-1. Of the 155 isolates of the genus Fusarium, 4 species of Fusarium were isolated (Table 3). The most frequently isolated species in all the 54 positive samples was found to be F. verticillioides. The other Fusaria isolated included F. pallidoroseum, F. oxysporum and F. solani.
All the Fusarium species found were determined to belong to some section from the infrageneric point of view. F. verticillioides belonged to section Liseola (Saccardo) Nirenberg, F. oxysporum belonged to section Elegans (Schlecht: Fr.), F. pallidoroseum belonged to section Arthrosporiella (Berkeley and Ravenel) and F. solani belonged to section Martiella (Martius) Appel and Wollenweber emend. Snyder and Hansen.
Fusarium verticillioides was most frequently isolated from all positive samples, i.e., in 88 samples. This represented a part of 89.09% (in total 330 isolates). The colonies on PDA were creamish to peach to vinaceous on the obverse and pale cream to salmon to violet or blue on the reverse (Fig. 1A). This taxon was characterized by mostly zero-septate, clavate microconidia with a flat base produced on monophialides in chains (Fig. 1B) in the aerial mycelium. The macroconidia were observed rarely which were mostly 3-septate in some isolates.
| Table 1: | Per cent incidence of F. verticillioides in animal feeds collected from different districts of Karnataka |
| Table 2: | Per cent incidence of Fusarium species in poultry feeds collected from different districts of Karnataka. |
| Table 3: | Frequency of fusarium species in poultry feed mixtures |
Fusarium species have been one of the most economically important groups of fungi. Reducing the realization value of cereals as food, feed and grain cereal, toxins produced by the fungi cause chronic and acute poisoning and allergic signs both to animals and humans. Therefore, throughout the world great attention is paid to investigating Fusarium species and elaborating means for controlling them. Contamination of cereals with Fusarium toxins is a global problem, occurring in Europe, the Americas, Asia and Australia (Placinta et al., 1999).
Many strains of F. verticillioides, isolated not only from corn and feeds but also from other substrates such as sorghum and millet, have been shown to produce fumonisins (Nelson et al., 1991; Norred et al., 1993). Since the number of F. verticillioides isolated from poultry feed mixtures and animal feedstuffs were very high, it poses a potential toxigenicity in these samples from the region of Karnataka, India.
| Fig. 1: | F. verticillioides: (A) Colony on PDA after 7 days of incubation and (B) Microconidia in chains on monophialides under 450X |
Studies conducted in the region of Karnataka have reported the occurrence of potential fumonisin-producing Fusarium species (Sreenivasa et al., 2006) from freshly harvested maize samples. In India, Janardhana et al. (1999) has reported mycotoxin contamination in maize grains. Nevertheless, it is corn that is used as one of the major ingredients in the production of poultry feed mixtures as well as a variety of animal feedstuffs like maize meal, maize pellets, maize powder, etc. This is evident from our investigation where, maize pellets showed a high incidence of Fusarium species whose major component is corn. F. verticillioides, the major species isolated in our investigation is also the most prolific fumonisin-producing fungus.
Sixteen out of the thirty-three samples of poultry mixed feeds collected from the region of Nitra, Slovakia, assayed for the incidence of Fusarium species revealed that F. proliferatum was the most frequently isolated species (Labuda et al., 2003).
However, samples assayed by us recorded F. verticillioides as the most frequently isolated species. Pieckova and Jesenska (2001) has reported the production fumonisin B1 by strains of F. verticillioides from maize products in Slovakia. One of the major concerns about fumonisins in animals is that it has been involved in Equine Leukoencephalomalacia (ELEM) (Thiel et al., 1991), pulmonary oedema in swine and toxic effects in poultry. Toxicoses have been observed in at least two species (Equidae and swine) and are suspected of being caused by consumption of moldy feeds. Studies to determine whether F. verticillioides contamination and fumonisins might cause disease in other farm animals, especially poultry, are being conducted by several investigators. Mycotoxicoses arising from the consumption of food containing ingredients contaminated with mycotoxins has been a major problem for poultry producers in many countries (Bhat et al., 1997; Prathapkumar et al., 1997). In India, aflatoxicosis has been well recognized as a major problem in poultry mainly because of the popularity of maize (Gutema et al., 2000) and sorghum as a dietary ingredient. Poultry are apparently more resistant to fumonisins than are swine and equines. In India corn and sorghum are used as bulk ingredients in the production of animal as well as poultry feeds. Also sunflower seed cake and groundnut seed cake are used in the production of poultry feed mixtures. Hence, the chances of Fusarium contamination in such feeds can be expected beyond doubt. The occurrence of F. verticillioides species in animal and poultry feed mixtures in India, can pose a threat to animal and poultry health (Prathapkumar et al., 1997). The fusaria could thus be a potential source of Fusarium mycotoxins and in turn the animal and poultry feed mixtures harboring such fusaria. This study warrants the need for analyzing the samples for Fusarium mycotoxins, especially fumonisins and also to design effective management strategies to prevent the occurrence Fusarium and fumonisin contamination in cereals widely used in production of animal and poultry feeds.
CONCLUSIONS
The study highlights a potential risk of animal and poultry feedstuffs getting contaminated with hazardous toxic compounds, thus making it necessary for further analysis and continual monitoring and evaluation of such feeds before they reach the consumer for animal and poultry nutrition, respectively.
ACKNOWLEDGMENT
This research was financially supported by University Grant Commission, Govt. of India, Project No. No. F.3-15/2004 (SR) dated 12.01.2004 is gratefully acknowledged.