Whereas an E. praecox infection is generally considered to produce little pathology, an infection with E. acervulina and E. mitis can result in mild enteritis followed by fluid loss and the malabsorption of nutrients. In more severe cases, inflammation of the intestinal wall with local bleeding (haemorrhages) and sloughing of epithelia (E. brunetti, E. maxima) or complete villar destruction resulting in extensive haemorrhage and death (E. necatrix, E. tenella) is seen in infected chickens1,2. Most of the highly pathogenic species invade the lower parts of the digestive tract. The life cycle of Eimeria is relatively short, from four to six days, and consists of two developmental stages; exogenous (in faecal matter) and endogenous (in host digestive tract). The exogenous stage starts after the release of unsporulated (non-infective) oocysts in the faeces. Sporulation of the oocyst occurs in the faeces and is encouraged by the right temperature, humidity and aeration (access to oxygen). In the sporulated oocyst, so-called sporozoites are formed and, from that point on, the oocysts are considered infectious. Sporulated oocysts are remarkably hard and protect the parasite from desiccation and chemical disinfectants, ensuring long-term survival in a poultry house environment1,2,3. The endogenous stage starts after ingestion of the infective oocysts by the chicken. In the micro-environment of the gizzard, sporozoites are released from the oocyst. Further down the digestive tract, the sporozoites invade and destroy epithelial cells and start the highly efficient reproduction cycle. This involves several rounds of asexual reproduction, followed by sexual differentiation, fertilisation and the shedding of unsporulated oocysts. The highly efficient copious reproduction of Eimeria species in the digestive tract and perfect survival strategy (sporulation) in faecal matter increase the chances of infection in avian flocks2 .
Vaccinations and dietary treatments
Good husbandry helps to reduce the risk of transmitting the coccidiosis-causing parasites. Additional treatments are essential, especially during the more delicate endogenous stage in the host digestive tract. Two categories of anticoccidial drugs are applied, i.e. ionophorous compounds (ionophores) and synthetic drugs (chemicals). Generally, ionophores cause death (coccidiocidal) to the parasite by interfering with the passage of ions across the cell membrane, whereas chemicals prevent the replication and growth (coccidiostatic) by inhibiting different biochemical pathways of the parasite1 . To reduce the emergence of drug-resistant strains, shuttle and rotation programmes are widely used2 . Still, the demand for alternative methods, such as vaccinations and dietary treatments, is increasing due to pressure from governmental agencies and consumers to ban the use of drugs in animals intended for human consumption. Moreover, coccidiosis resistance to anticoccidials leads to economic losses for the industry.
Use of vaccines
Besides the number of ingested sporulated oocysts, the severity of coccidiosis highly depends on there being immunological memory for the pathogen. As early as 1923, Johnson published the first articles showing that resistance to a dose of oocysts was not age-dependent, but relied on earlier exposure to the parasite2 . Today, we still make use of this knowledge through the application of live (attenuated) vaccines. From a dietary point of view, different strategies can be used to fight coccidiosis. Some products act anti-microbially against Eimeria-specific species, e.g. essential oils and herbal extracts. Other products beneficially modulate the immune status of the chicken, whereas prebiotics and probiotics improve microflora to reduce the chance of secondary infections (e.g. C. perfringens). Damaged intestinal tissue benefits from additional antioxidants to reduce the vicious cycle of oxidative stress caused by damaged cells1 . Other products improve intestinal protection and mucosal healing, e.g. betaine, butyrate or threonine4,5,6.
Necrotic enteritis lesions
Coccidiosis in poultry often pre-exists or occurs concurrently with field outbreaks of necrotic enteritis7 . The causative agent of necrotic enteritis is Clostridium perfringens, a gram-positive spore forming anaerobic bacteria commonly found in soil, dust, faeces, feed, poultry litter and intestinal contents8 . Necrotic enteritis has long been controlled by using antibiotic growth promoters (AGPs) in feed. Nonetheless, in January 2006, the ban on dietary AGPs took effect in Europe and the use of AGPs is under discussion on other continents. Necrotic enteritis has emerged as a common broiler disease worldwide9,10. The conventional therapeutic antimicrobial agents and anti-coccidial drugs, which not only exert an effect against Eimeria spp but also against C. perfringens, are currently used to control necrotic enteritis8 . But this approach conflicts with the objective of reducing the use of antibiotics in animal production. Understanding the pathology and predisposing factors of necrotic enteritis is helpful in searching for preventive alternatives. Necrotic enteritis usually occurs three to four weeks after hatching11. The necrotic lesions are mainly restricted to the small intestine and the infection can result in an acute clinical disease or is present in a subclinical condition. In clinical cases, there is increased flock mortality during the last weeks of rearing, often without premonitory signs. The disease is acute, with death occurring within one to two hours, and mortality rates can rise up to 50%. Broiler flocks with subclinical necrotic enteritis do not display clear clinical signs and usually there is no peak mortality. Chronical damage to the intestinal mucosa leads to poor digestion and absorption of nutrients, resulting in reduced weight gain and increased feed conversion9,10,12. In subclinical conditions, feed intake can be reduced by 35% during the infectious period13. In certain cases, intestinal damage can allow C. perfringens to reach the bile duct and portal blood stream. Colonisation of C. perfringens in the liver results in cholangiohepatitis, lesions to the liver, which are finally found at the slaughter line12,14. Although clinical outbreaks of necrotic enteritis may cause high levels of mortality, the subclinical form is economically more important, as it often stays undetected in the broiler flock. Hampered growth and an increased number of condemnations at the slaughter line causes significant economic losses to the poultry farmer. The true economic impact of necrotic enteritis is not from birds that die from infection, but rather those that suffer from disease and survive the subclinical form (12).
Available nutrients allow proliferation
Clostridium perfringens is commonly found in the normal healthy avian gut microbiota. The virulence of a strain depends on its nature. Strains of C. perfringens are classified into five different types (A to E) on the basis of the production of four major toxins. Clostridium type A is associated with necrotic enteritis in broilers, although this type is also found in the digestive tract of healthy poultry9 . As necrotic enteritis is a multifactorial disease, more predisposing factors are needed for its development (Figure 2)10. High levels of (animal) protein or poorly digestible protein sources are associated with a higher risk of necrotic enteritis, as undigested protein is a growth substrate for pathogenic bacteria such as C. perfringens. Wheat, rye, oats and barley are more often associated with necrotic enteritis, due to higher levels of indigestible, water soluble, non-starch polysaccharides, whereas maize is not. Besides the choice of raw materials, particle size also seems to influence gut health. Feed containing many small and some largesized particles is more predisposed to necrotic enteritis than feed containing uniform particles7,10,15. Changes in the feeding regime (moving from starter to grower diets), other diseases and increased stocking density increase stress in the flock and suppress the immunological status of the chickens, making them more sensitive to necrotic enteritis infection12.
Coccidiosis infection is an important predisposing factor, as the mucosal damage caused by Eimeria provides a favourable environment for C. perfringens to proliferate. Synergism between coccidiosis-causing protozoan Eimeria and necrotic enteritis-inducing C. perfringens is shown in Figure 3. It is interesting to note that C. perfringens needs high-quality protein, as it requires 13 essential amino acids12. By killing epithelial cells, Eimeria induces leakage of plasma proteins (A) and the coccidial infection enhances mucus production (B). Both effects provide an increased nutrient availability for C. perfringens to grow12. The virulence of the C. perfringens strain depends mainly on four major factors: its ability to produce bacteriocins, bacterial collagenolytic enzymes, toxins and its ability to adhere to the gut wall (Figure 2).