TOXIN GENOTYPES OF Clostridium perfringens IN ANIMAL FEED AND THEIR ROLE IN THE ETHIOLOGY OF ENTEROTOXEMIA IN DOMESTIC ANIMALS

Clostridium perfringens is a Gram-positive, endospore-forming, anaerobic rod, ubiquitous in nature. C. perfringens strains can produce about 17 toxins. Many of them can lead to miscellaneous diseases, among which the enteric ailment may be the most common and is of utmost importance. In the present work 34 strains of C. perfringens isolated from feed and one from a cow suspected to have died of clostridial infection were subjected to molecular analysis. In order to detect the genotypes, the following genes coding for toxins were targetted: cpa, cpb, cpb2, cpe, etx and iap. The multiplex PCR assay revealed that all C. perfringens isolates from animal feed were of type A and 2-toxinogenic type A strains, possessing only the cpa (n=21), or both the cpa and the cpb2 genes (n=13). The importance of C. perfringens toxins  and -2 in the pathogenesis of enterotoxemia is discussed and the regulation on the detection of this bacteria in animal feed questioned. The use of PCR in practise could enable the toxin-genotyping of C. perfringens isolates and, thus, provide a real basis for the establishment of maximum acceptable limits of this bacteria in feed.


INTRODUCTION
Clostridium perfringens is a Gram-positive, non-motile, endospore-forming, anaerobic (relatively oxygen-tolerant) bacteria, ubiquitous in nature (found in soil, dust, sewage, and fresh and marine water) and a common inhabitant of the intestinal tract in both humans and homeothermic animals (Songer, 1996;Brynestad and Granum, 2002). The species represents a very heterogeneous group with respect to metabolic bioproducts, toxins and pathogenicity. C. perfringens strains can pro-duce about 17 toxins (Uzal et al., 2015;Freedman et al., 2016), four of which (α, β, ε and ι) are major factors of virulence. Based on the capability to produce them, C. perfringens is classified into five toxotypes: A, B, C, D and E (Hatheway, 1990; Sawires and Songer, 2006). Two other major toxins are enterotoxin (CPE) and -2 toxin, both possibly produced by all types of C. perfringens (Ata et al., 2013). Each toxotype may cause different diseases in animals, including acute enteritis and fatal enterotoxemia (Baums et al., 2004;Ata et al., 2013;Lyhs et al., 2013). Some varieties within types A, B and C produce certain combinations of antigens or toxins associated with defined diseases or syndromes (Ata et al., 2013;Uzal et al., 2010Uzal et al., , 2014. Their quantities vary greatly between individual strains (Niilo, 1980). C. perfringens may enter feed via contaminated raw ingredients, or by secondary contamination during processing, storage and dispersal of final products. Feed contamination depends on its composition, the hygiene during production and the storage conditions, and is directly proportional to the levels of soil and faecal contamination (Wojdat et al., 2006).
In the Republic of Serbia, in compliance with regulations (Regulations on the quality of animal feed), feed samples of 50 g must not contain any C. perfringens. For food it is stated in The EFSA Journal (EFSA, 2005) that "C. perfringens is commonly present in foods and ingredients, occasionally at hundreds per gram, and microbiological testing for C. perfringens has limited value in ensuring food safety, because the organism is so common in or on foods that a positive result means little, unless very high numbers are present". In spite of being notorious for extremely toxigenic potential, not all of C. perfringens strains are capable of producing illness in neither animals nor humans.
Experimental researh has confirmed that the ingestion of C. perfringens will not lead to sickness per se, because the majority of bacteria consumed with food are destroyed by hydrochloric acid in the stomach. Moreover, C. perfringens does not exhibit adherence and invasive properties towards healthy intestinal mucosa. The development of subclinical or clinical clostridiosis in domestic animals always results from complicated interactions between the virulence of the bacteria (which is highly variable within this species), immune status of the host and the influence of a variety of non-specific factors associated with the farm management.
Gut microbiota disturbances (most frequently resulting from antibiotic therapy), management-related stress, sudden chan-ges in diet, and overeating and/or voraciousness, especially on high-protein and energy-rich foods, are predisposing factors for the development of enteritis and enetrotoxemia (Collier et al., 2008;Timbermont et al., 2011;Uzal et al., 2015).
For all these reasons, without the identification of toxin genotypes and the levels of feed contamination it is difficult to assess the health risk C. perfringens in feed poses to animals. Nonetheless, toxin genotyping of C. perfringens strains isolated from food stuffs and animal feed is not part of routine laboratory practice in Serbia.
In this work C. perfringens isolates (n=34) from food intended for animals were subjected to multiplex PCR essay in order to detect genes: cpa ( toxin), cpb (), cpb2 (2), etx (), iap () and cpe (enterotoxin). Further, the importance of those findings are discussed from the standpoint of health risk to food-producing animals and, indirectly, to humans, consumers of animal products. The results of such investigations can be the basis for risk assessment and determination of critical limits of the presence of C. perfringens in animal feed.

MATERIALS AND METHODS
C. perfringens isolates. C. perfringens was isolated from feed samples according to the standard SRPS ISO 7937:2010. The species identification was based on: (1) the characteristics of the colonies grown on agar with the addition of 5% sheep blood and incubated in anaerobic conditions (GasPak EZ, Becton Dickinson and Company, USA), (2) synergistic haemolysis with Streptococcus agalactiae (reverse CAMP test), and (3) biochemical properties: catalase test (negative), fermentation of glucose and lactose and production of lecithinase. Thirty-four isolates of C. perfringens were subjects of genotyping. Their origin is presented in Table  2. In addition, one isolate from the organs of a cow which was suspected to have died of enterotoxemia was assessed: the organs were sent to the laboratory with the feed that was allegedly the source of the infection.

DNA extraction and PCR amplification
From C. perfringens isolates incubated overnight on blood agar suspensions in PBS were made and turbidity adjusted to 3.5 McFarland. One milliliter was transferred to an Eppendorf tube and boiled in water for 15 minutes. The tubes were cooled on ice and centrifuged for 8 min at 11,000 x g. The supernatant obtained (100 L) was preserved in a freezer at -20 C until being processed further. The multiplex PCR assay (mPCR) was performed according to the protocol described by Baums et al. (2004). Six sets of oligonucleotide primers were used ( Table 1). The mPCR method was carried out using the "HotStar Taq Master Mix Kit" (Qiagen, Hilden, Germany), with small modification of manufacturer's instructions. Briefly, the amplification reaction was carried out in a volume of 25 μl containing 3 μl of DNA sample, 12.5 μl of master mix and 25 pmol of each primer. C. perfringens ATCC 13124 was used as the positive and C. sporogenes ATCC 19404 as the negative control.
The cycling conditions (Thermocycler Gradient, Eppendorf, Germany) were the following: initial denaturation at 95 C for 2 min 30s, 40 cycles of 1 min at 95 C, 1 min at 55 C and 1 min at 72 C, which was followed by the final extension for 10 min at 72 C. Ten microlitres of the PCR products were separated by electrophoresis on a 1.5% agarose gel, stained with ethidium bromide (0.25 g/ml) and documented with a gel documentation system (GelDoc-It 2 Imager, UVP, Cambridge, UK).

RESULTS
All of the C. perfringens isolates formed small translucent colonies surrounded by the zone of double haemolysis on blood agar ( Figure 1A). Clear zone of haemolysis results from the theta () toxin, whilst the -toxin is responsible for incomplete haemolysis. The production of -toxin in all isolates was confirmed with the positive reverse CAMP test ( Figure 1B).
The multiplex PCR assay revealed the presence of specific amplicons: 900-bp long, which are characteristic of cpa gene, 396 bp of etx, and 200 bp of the cpb2 gene encoding for C. perfringens toxins (Figures 2, 3 and Table 2). All C. perfringens isolates derived from feed were identified as type A and 2-toxinogenic type A strains, since they possessed only the cpa (n=21), or the cpa and the cpb2 genes (n=13). In neither isolate from feed genes coding for beta (cpb), epsilon (etx) and iota toxin (iap) were detected.   Gene for the enterotoxin (cpe) was detected in only one strain, which was isolated from the cow's organs.

DISCUSSION
C. perfringens is notorious for being one of the common contaminants of various foodstuffs, which was also proven in this research conducted on several types of animal feed (Kanakaraj et  In Poland 334 C. perfringens isolates from animal feed stuffs were processed with multiplex PCR to detect six genes coding for toxins. Most of the isolates, 50%, possessed genes coding for both  and β2 toxins, whilst the gene responsible for  toxin only was detected in 46.4% isolates, which means that the vast majority of the strains were C. perfringens type A. C. perfringens types E, C, (including the one with a β2 toxin gene) and type D capable of producing β2 toxin made up for less than 4% of the isolates in total (each present in less than 1% of the isolates), whilst C. perfringens type B was not detected (Wojdat et al., 2006).
C. perfringens type A is the most common of all C. perfringens types, and is part of the normal gut microbiome in humans and animals. Of all major lethal toxins, type A produces only the -toxin, common to all C. perfringens toxotypes (Uzal et al., 2010). The -toxin is an enzyme, a phospholipase which hydrolyzes lecithin (Niilo, 1980;Yoo et al., 1997). Because the membranes of the majority mammalian cells are composed of lipoproteins containing lecithin, its destruction leads to necrosis, hemolysis, or even death, depending on the tissue(s) involved. The -toxin is essential for the pathogenesis of gas gangrene and its production is considered a housekeeping capacity of all C. perfringens strains. However, the role of this microorganism and its major -toxin in natural intestinal disease remains controversial and poorly documented (Uzal et al., 2010). C. perfringens type A is even considered to be the most confusing organism in respect to its pathogenicity (Niilo, 1980;Ata et al., 2013).
C. perfringens type A has been reported to cause enterotoxemia in sheep and calves, sometimes even leading to sudden deaths. However, most of the data were not proven and based mainly on bacterial isolation and clinical signs, which were unspecific. Alpha toxin is present in the gut content of many clinically healthy animals. Thus, its detection in the intestinal content of affected animals is irrelevant for the diagnosis of the disease (Uzal et al., 2010). In addition, C. perfringens type A can be readily isolated from samples taken soon after death, can be grown easily and may mask other bacteria, implying that it had been the only causative agent. Researchers failed to produce acute enterotoxemia in calves and sheep by intraduodenal administration of C. perfringens type A. Administered intravenously, the toxin led to death only in relatively large doses applied within a short time, whilst slow application resulted in milder effects, probably due to rapid elimination. In sum, it seems unlikely that α-toxin can be produced in the intestines in high quantities and absorbed rapidly enough to result in fatal toxemia (Niilo, 1980). One of the difficulties in the understanding of the pathogenesis and diagnosis of enteric disease caused by C. perfringens is based on the fact that most of its types can frequently be found in the intestines of healthy animals. Simple isolation of C. perfringens is therefore of no use in diagnostic of enteric infections (Uzal et al., 2015).The intestines of birds suffering NE contain large numbers of C. perfringens, up to 10 6 or 10 8 CFU/g of intestinal contents (Cooper and Songer, 2009), whereas in healthy broilers the counts usually range up to 10 2 , or 10 3 CFU/g (Baba et al., 1997;Kukier et al., 2010). For epizootical analysis of animal diseases caused by C. perfringens, strains isolated from affected birds and the feed they had consumed should be compared. First, the toxotype and subtype is to be detected (with PCR), which is followed by the confirmation of identical DNA profiles (with pulsed-field gel electrophoresis) (Kukier et al., 2010). However, additional research may detect some other anaerobes in feed, which may also conduce to diseases. PCR analysis may detect that healthy birds can carry 2-5 genotypes of C. perfringens type A, contrary to those affected, which are colonized by a single genetic type other than that found in diseased birds (Cooper and Songer, 2009).
The answer to the question whether C. perfringens is a general foodborne pathogen is immensely complicated. In its toxigenic behaviour type A can be subdivided into two varieties. The "classical" variety, characterized mainly by -toxin production, is associated with gas gangrene, traumatic infections and the normal intestinal tract. In the beginning, -toxin was considered to play the main role in human food poisoning and enteritis, but another variety of type A, which is producing the enterotoxin (CPE), was later determined. The enterotoxin was first isolated in 1970 (Brynestad and Granum, 2002 . CPE is produced in the small intestine after ingestion of at least 10 7 C. perfringens cells (Brynestad and Granum, 2002).
Molecular detection of genes encoding for enterotoxins is extremely important in the inspection of animal-derived foodstuffs and is more reliable than conventional enterotoxin detection; they are all intended for the prevention of the entrance of these strains into the food chain (Kanakaraj et al., 1998). In this research, the gene coding for enterotoxin was detected only in one C. perfringens strain isolated from a dead cow, but not in the feed mixture which the animal had been fed on. This result implies that animal feed is irrelevant to the entrance of cpe-positive strains of C. perfringens to the food chain. Previously, the use of toxin genotyping of C. perfringens from swine feed sampled on different farms (n=60) found no isolates positive for cpe (Kanakaraj et al., 1998). In addition, molecular typing of isolates obtained from swine denied that this species was a source of cpe-positive strains of C. perfringens (Kanakaraj et al., 1998).
Gene coding for -2 toxin (cpb2) was confirmed in 13 C. perfringens isolates: in those originating from feed intended for poultry (n=4), pigs (n=4) and cows (n=4), and from a praemix (n=1). This toxotype might be important for the development of enteric diseases in pigs and cattle. A significant correlation (82%) between C. perfringens possessing the 2-toxin gene and diarrhoea in piglets was identified, suggesting that 2-toxin may play a key role in the pathogenesis of the disease (Garmory et al., 2000). More than 85% of isolates from cases of porcine enteritis and 91.8% isolates of newborn pig enetiritis were positive for the cpb2 gene (Bueschel et al., 2003). However, cpb2-positive strains can be found in both healthy and diseased chickens and its presence does not correlate with disease (Keyburn et al., 2008). Pulsed-field gelelectrophoresis performed on C. perfringens isolated from turkeys with NE and from healthy birds, in all of them detected -toxin, and in one both  and 2 toxins, which indicates that the β2 toxin is not an important virulence factor in the development of NE (Lyhs et al., 2013). Beta-2 toxin has been associated with porcine, equine and bovine gastroenteritis (Bueschel et al., 2003;Van Immerseel et al., 2004).
In type A strains isolated from various animals, food products and environment the prevalence of the cpb2 gene is high (Bueschel et al., 2003;Garmory, et al. 2000; Mueller-Spitz et al., 2010). The gene is plasmid-borne, at least in some strains (Shimizu et al., 2002), which implies its mobility and the possibility of transfer among strains of C. perfringens.
In this research all animal feed samples from which C. perfringens was isolated the spore numbers were less than 100 CFU/g. In similar trials the numbers in feeds did not exceed 10 2 CFU/g (Kukier et al., 2010) or 10 3 CFU/g (Wojdat et al., 2006). Thus, it is reasonable to raise the question why the numbers of C. perfringens in feed are to be assessed given that the Regulation orders that feed be condemned if this species is even detected. Estimating the numbers would be sound if maximum allowed numbers per gram had been established. The PCR test ensures the detection of all genes considered to be important virulence factors in C. perfringens-mediated enteritis or enterotoxaemia and thus provides a useful and reliable tool for C. perfringens genotyping in routine veterinary diagnostics (Baums et al., 2004). The mPCR assay is recommended for risk assessment of C. perfringens toxotypes isolated from animal feed. The obtained results can help to establish microbiological criteria for critical limits of the presence of C. perfringens in feed (Wojdat et al., 2006).