Fungistatic and mycostatic activity

The growth of fungi in poultry feed is a common problem. Various species of fungi may colonize plant tissue before or after harvest, the two primary factors that influence fungal growth being temperature and moisture (CAST, 2003). Damaged or broken grains and insect infestation can also facilitate contamination, and improperly stored grain can present ideal conditions for fungal growth. While the fungi itself is not necessarily considered dangerous, it can cause a decreased nutrient value of the feed, decreased palatability or acceptability, possible respiratory problems, and toxicosis due to the ingestion of mycotoxins produced by the organism. The problem of mycotoxin production is a serious consideration globally when preparing feed for animal consumption. Mycotoxins are toxic fungal secondary metabolites that, when consumed, can cause chronic toxicoses resulting in inefficient production or, at high doses, acute toxicoses resulting in disease or death, not to mention economic losses (Nichols, Reference NICHOLS1983; Shane, Reference SHANE1994). The majority of mycotoxins that are considered to be of most importance to animal agriculture are produced by three genera of fungi. These include Aspergillus, Penicillium, and Fusarium (CAST, 2003), with aflatoxicosis, fusariotoxicosis, and ochratoxicosis occurring most commonly in poultry.

Many methods have been employed to reduce the occurrence of mycotoxins in feed, including screening for moulds, proper management in feed production facilities, and the inclusion of products that bind mycotoxins in feed such as hydrated sodium calcium aluminosilicate (HSCAS) (Phillips et al., Reference PHILLIPS, KUBENA, HARVEY, TAYLOR and HEIDELBAUGH1988). The use of commercial mould inhibitors is a popular method for controlling mould growth in feed. Examples of these include mixtures of organic acids such as propionic acid, acetic acid, sorbic acid and salts of organic acids such as calcium propionate. When distributed evenly and completely throughout the feed, some mould inhibitors can be effective in preventing the growth of moulds, although many will not kill the mould or prevent mycotoxins in the feed. These inhibitors may have to be reapplied after a period of time, as the concentration decreases after the initial application. In addition, some fungal inhibitors, such as propionic acid, can lead to corrosion of metal equipment. Others, including anhydrous ammonia, can be potentially hazardous to the handler, and may lead to the corrosion of electrical components. Many of these compounds have been shown to damage the seeds ability to germinate, resulting in treated grains only suitable for feed, as it has lost its viability as seed grain. One must also keep in mind that organic acids may only work well in certain feedstuffs, but are not effective in others. Furthermore, there is little research data available on the relative efficacy of these acids at various concentrations and, therefore, the industry continues to rely almost solely upon research conducted in the early 1970s (Pelhate, Reference PELHATE1973).

HRC is a compound that can be incorporated as a mould inhibitor into the feed itself as a means of suppressing fungal growth and the accompanying production of mycotoxins. This can be very useful in those many countries where poor storage conditions are prevalent. As early as the 1920's (Stearn and Stearn, Reference STEARN and STEARN1929) this compound was shown to have antifungal effects, and has been added to feeds in the form of commercial products for many years. Chen and Day, (Reference CHEN and DAY1974) conducted a study comparing HRC as a fungal inhibitor to other chemicals commonly used as fungistats. Fungal species tested were Aspergillus flavus, Aspergillus fumigatus, Candida albicans, Fusarium moniliforme, and Penicillium camemberti. In general, 0.05% GV-11 (a commercially manufactured HRC compound, Naremco, Inc.) was effective in inhibiting the growth of the tested mould species, and exerted a greater controlling effect than 0.05% of sodium propionate, calcium propionate, and propylene glycol. HRC was also shown to be extremely effective in controlling the growth of Candida albicans, a pathogenic yeast-like fungus that causes crop mycosis in poultry (Balasubramaniam and Sukumar, Reference BALASUBRAMANIAM and SUKUMAR2007).

Chen and Day (Reference CHEN and DAY1974) reported superior efficacy of GV – 11 compared to two other products containing propionic acid or nystatin. Higgins and Brinkhaus (Reference HIGGINS and BRINKHAUS1999) reported that organic acid fungal inhibitors such as propionic acid and butyric acid were required in concentrations of 0.1% and 0.2% to inhibit Aspergillus spp. or Fusarium spp., respectively, with complete inhibition occurring at concentrations up to 0.35%. Likewise, more recent data in our laboratory (Table 2) indicates that corn grain added with 10% water and supplemented with either 0.05% or 0.1% GV-11 resulted in less than a million CFU/g of moulds of different species as compared to a control. Corn that was not treated with water or GV-11 generated two log CFU/g (100 x) less moulds. Corn treated with GV-11 had three log-fold (1,000 x) less mould than the control corn and 10 x less than the untreated corn.

Table 2

Mould growth on corn with or without the addition of HRC.

* nb: counts are reported as CFU per g of corn

An additional benefit of using HRC involves the pelleting process of poultry feed. Most feed in the poultry industry is pelleted with the exception of starter period feeds, which are usually manufactured in the form of mash or crumbles. Chen and Day (Reference CHEN and DAY1989) demonstrated that the activity of HRC was enhanced by a short period of heating meant to mimic the process of pelleting. Some other important considerations taken into account in this study include particle size of both the inhibitor and the feed ingredient. In general, greater inhibition was achieved when the particle size of the corn meal in the experiment and the HRC was decreased. This is most likely due to better dispersion, and therefore better physical contact with fungus in the feedstuff.

Bactericidal activity

The bactericidal activity of HRC has long been studied and it is known that gram-positive bacteria are not only killed by the compound, but also will not grow on HRC media, while most gram-negative bacteria are unaffected (Churchman, Reference CHURCHMAN1921; Churchman, Reference CHURCHMAN1923). Gram-positive bacteria include staphylococci, streptococci, clostridia, and species of the genus Bacillus. Graham and Nelson (Reference GRAHAM and NELSON1953) reported that HRC can inhibit the growth of Streptococcus lactis by increasing the length of the lag phase. Additionally, Tolba and Eskarous (1962) demonstrated that HRC has the ability to inhibit the haemagglutination activity and infectivity to chick embryos of all strains of Newcastle disease and fowl-plague viruses at treatment concentration above 100 ppm. An increase in concentration up to 2000 ppm did not result in any toxic effects that compromised the viability of the embryos.

Residue and toxicity

In a study conducted by the Southern Research Institute (Reference SOUTHERN RESEARCH INSTITUTE1973a), researchers were unable to detect any HRC (including colour) accumulation in the edible portions of broilers that had consumed 35, 96, or 300 mg/kg body weight of gentian violet after eighty days of feeding. Dietary treatments included a control diet (no GV-11), 500 g/ton of feed, 1.5 kg/ton of feed, and 4.5 kg/ton of feed (normal inclusion rates for GV-11 are between 0.5 to 1 kg/MT final feed.) Another study conducted by the same group (1973b) determined that single oral doses of 150 and 300 mg/kg body weight of HRC were lethal to adult chickens, and a dose of 2.5–5.0 g/kg body weight GV-11 was also lethal. The conclusion was made that the single oral dose LD₅₀ for GV-11 is less than 5.0 g/kg body weight but slightly more than 2.5 g/kg body weight.