The pathogenicity of different Eimeria ranges from moderate to severe (36), some species may cause loss of fluids and a decrease in nutrient absorption (E. acervulina and E. mitis), swelling of the intestinal wall with petechiae and loosening of the epithelium (E. brunetti and E. maxima) or complete destruction of villi, producing hemorrhages and death (E. necatrix and E. tenella), each species causes recognizable and distinct signs of coccidiosis, independent of the other species (8, 53).

Coccidial infections are self-limiting and depend largely on the number of sporulated oocysts ingested (6). Several studies have shown that there is an optimal dose, such that the reproductive potential of the parasite is met and they efficiently replicate within the epithelial cells. Very high doses of ingestion can cause the so-called “crowding effect,” interrupting the continuity of the parasite's life cycle, while still generating intestinal damage (54–56). For example, Williams in 2001 (55) was able to characterize the reproductive potential of each species of Eimeria under experimental conditions, using infective doses of 903, 16, 39, 14, 16, 16, or 72 sporulated oocysts, of E. acervulina, E. brunetti, E. maxima, E. mitis, E. necatrix, E. praecox or E. tenella, respectively.

The host is a pivotal factor of Eimeria infections in poultry (6). Some Eimerian parasites are highly immunogenic in chickens, and primary infections can stimulate protective immunity to subsequent challenge by the homologous parasite (57). Lillehoj in (58) used two chicken lines SC (B²B²) and FP (B¹⁵B²¹), to demonstrate that age and host genetic background can affect the outcome of coccidial infections. These two lines were subjected to an experimental infection with different doses of sporulated oocysts of Eimeria tenella, where is was found that in a reinfection process at different ages, the FP line was more resistant to Eimeria than the SC line when the primary innoculation contained a high dose of oocysts. Further, they determined that older animals demonstrated total immunity to Eimeria.

Additionally, some publications suggest that host sex influences the prevalence of coccidiosis; for example, Hadas et al. (59) and Wondimu et al. (60) report a prevalence of coccidiosis in farms of Gondar Town, Ethiopia that is relatively higher in male (44.3–43.6 percent) than female chicken (42.4–41.2 percent), but no significant statistical difference was found in either study.

There are specific factors that jeopardize and increase the spread of the parasite, including inadequate biosecurity protocols and poor hygiene of both personnel and equipment (60, 61). Sanitization plays a major role in reducing the dissemination of the parasite (62), as the most frequent mode of transmission of oocysts is through mechanical vectors such as movement of personnel or equipment between farms, and the presence of rodents and insects such as flies and beetles (6, 63).

Correct identification of Eimeria species is important for the diagnosis and control of the disease (64) and from a commercial standpoint, a diagnosis of coccidiosis is required when the gross lesions are evident (6, 49). Classical methods for the evaluation of Eimeria infections include macroscopic diagnosis with observation of clinical signs in infected animals, the location and appearance of gross lesions during necropsy; and microscopic diagnosis, which focuses on evaluating the size and shape of oocysts (Figure 2) (49). Sometimes the evaluation of other developmental stages in microscopic smears is also included (65). In addition, when greater diagnostic precision is needed, molecular diagnostics can be included (35, 66, 67).

Coccidia invade the intestinal mucosa and induce a certain degree of epithelial cell damage and inflammation. Meronts, gamonts, and oocysts cause marked histological alterations of host intestinal epithelial cells over a short time period including distortion, rupture, separation from adjacent cells, and sloughing (68). Infected birds present with ruffled feathers and signs of depression or drowsiness (Figure 2). Additionally, feed and water consumption are decreased, and the feces may be watery, whitish and occasionally bloody (69). This results in dehydration, impaired weight gain, and in the absence of treatment, death (61). Additionally, there is the problem of malabsorption due to reduced brush border enzyme activity (70) and disruption of intestinal integrity (71).

Infection can cause other intestinal changes, as well; for example, an inoculation with E. acervulina and E. maxima oocysts increased the size and number of goblet cells along ileal crypts in broilers (72). Goblet cells represent an important defense mechanisms in the intestinal tract (73), secreting glycoproteins of high molecular weight called mucins (74). Mucins are the first line of defense against intestinal pathogens and act to protect the epithelium from pathogens and irritants in the intestinal lumen (75). Similarly, it has been reported that when E. tenella invades cecal epithelial cells, the cecum increases the rate of mucus production and promotes a protective phenotype as an immunological reaction against the parasite (76, 77). However, this increase in mucin production can also be harmful, promoting secondary colonization by other pathogens such as Clostridium perfringens (72, 78, 79), Salmonella and certain viruses like Marek's disease virus or infectious bursal disease virus (6, 80). This has the effect of further altering intestinal health by impairing metabolism and nutrient absorption (81).

For the evaluation of gross lesions, a standardized intestinal lesion scoring technique is used (82), which is based on giving a score on a scale of zero to four, with the goal of obtaining a numeric classification of the gross lesions caused by each Eimeria species (65, 67–83) (Figure 2). For this scoring system, the entire intestine of the bird must be evaluated, beginning with the duodenum. The mucus and serous membranes are examined to detect lesions, and a good light source (solar or lamp) is essential for reliable scoring (49). Table 3 summarizes the changes visible in the walls of infected organs by oocysts of E. acervulina, E. maxima and E. tenella and respective lesion scoring. Generally, a set number of birds per flock are assessed (between 5 and 6) and the individual scores are added for all Eimeria spp. (84). This is a laborious method, it can be subjective, and it needs experienced personnel to obtain an accurate outcome. However, it is still the most widely used diagnostic method (49).

Scrapings of the intestinal mucosa can be taken to evaluate the presence and shape of oocysts (65) or this count can be done using droppings (85). Typically, the intestinal lesion score is complemented with counts of oocysts per gram (OPG) of feces or poultry litter through the McMaster technique (86). It is believed that the correlation between lesion scoring and productive performance is stronger than the relationship between oocyst counts (OPG) and performance (49). Perhaps, oocyst shedding does not correlate well with decreased body weight gain or intestinal lesion scores because high doses of Eimeria can result in a crowding effect that reduces oocyst shedding while still causing significant damage to the intestine (55). Regardless, greater accuracy is needed to determine the level of intestinal lesions at which performance begins to be impacted, especially when subclinical conditions are present (49).

Recently, the Mini-FLOTAC (Figure 3) was developed as a new method for qualitative and quantitative diagnosis of infections by helminths and protozoans in several mammal hosts. This is a useful technique to process large amounts of samples rapidly at the laboratory or at the farm (87). The Mini-FLOTAC technique is based on flotation principles with saturated solutions, using a device with two components, the Fill-FLOTAC and reading chamber (86). The Fill-FLOTAC is a clear plastic container with a capacity of 70 mL, used to carry out the first four steps of the technique i.e., sample collection and weighing; homogenization; filtration; and filling of the chambers. The other component is the Mini-FLOTAC reading chamber, which has two components (the base and the disk with two reading chambers, 1 mL each, with ruled grids on the surface, which divide each chamber into 12 sections) and two accessories (the key and the microscope adaptor) (87).

The microscope reading is done at a magnification of 100x (86), up to 400x when there are small sized protozoa (87). This technique has become an alternative to the McMaster method, especially in cases where greater accuracy is needed (60), and has been used successfully in different species including goats and horses (88, 89). According to Bortoluzzi et al. (86), who compared the precision of the McMaster technique and the Mini-FLOTAC to quantify Eimeria maxima oocysts, the Mini-FLOTAC is a reliable and precise method of quantification for this species with the detection limit ranging between 100 and 500 oocysts per gram of excreta (OPG). Other works like Das et al. (90), found the Macmaster technique to be less sensitive than Mini-FLOTAC when the load was 50 OPG, whereas there was no difference observed when 100, 250, 300, 450, 625 and 1,250 OPG were present. Additionally, this study concludes that the McMaster method is faster than Mini-FLOTAC and both McMaster and Mini-FLOTAC underestimate true OPG counts.

In practical conditions, Eimeria infections are often caused by more than one species, with similar pathological characteristics, making diagnosis in the field difficult (64). This means that methodologies that can be more sensitive and less subjective are very important for correct diagnosis (91). Molecular biology techniques offer improved diagnostic precision in many cases. One example is polymerase chain reaction (PCR) based identification of Eimeria; the amplification of regions of the internal transcribed space 1 (ITS1) of the ribosomal DNA (92–94) is now a widely available technique. The ITS is a piece of non-functional RNA located between structural ribosomal RNAs (rRNA) on a common precursor transcript. This region of the genome contains several segments used for Eimeria spp. identification, including the 5' external transcribed sequence (5'ETS), 18S rRNA, ITS1, 5.8S rRNA, ITS2, 26S rRNA, and finally the 3'ETS. Because it is easy apply this technique with only small amounts of DNA and because there is a high degree of variation between closely related species, the ITS region is widely used in molecular phylogeny and taxonomy (91). Other molecular techniques that have been reported for the identification of Eimeria species have been Random Amplified Polymorphic DNA (RAPD) (95), Sequence Characterized Amplified Region (SCAR) markers (96, 97), quantitative PCR (7) and Loop-Mediated Isothermal Amplification (LAMP) (97, 98). Furthermore, multiplex PCR techniques for detecting the seven Eimeria species of interest have been described, which combine primers for each species in a single reaction (99). Given that this is a parasite with a wide epidemiological distribution and with reports of taxonomic variants throughout the world (100), continued study is necessary. Continued advances in Next Generation Sequencing techniques (NGS) are allowing for precise identification of emerging or region-specific Eimeria spp. as well as facilitating other avenues of research for control of coccidiosis (66, 67).

Since the 1950s, it has been common to raise broiler chickens and turkeys with feed anticoccidials. According to Agri Stats Inc. (Fort Wayne, IN), in the late 1990s, 99% of broiler chickens were raised with an anticoccidial drug in one or more phases, and this practice is still prevailing in many markets (102). However, trends in some markets are changing and today, some of the largest producers of broiler chickens in the world, like the USA, are raising up to 60% of broilers without anticoccidials (103).

Anticoccidials, based on their mode of action, may be divided into coccidiostats and coccidicides. Coccidiostats halt the development of the parasite, compromising its replication and growth, but their effect can be reversible, as removal from the diet can lead to the re-emergence of the disease. Coccidicides are characterized by killing or causing irreversible damage to the parasite (62).

Anticoccidials may also be classified into two categories according to their origin (10, 104): (1) synthetic compounds, which are produced by chemical synthesis and have a specific mode of action against the metabolism of the parasite (10); (2) polyether or ionophore antibiotics, which are produced by the fermentation of Streptomyces spp. or Actinomadura spp., which generally destroy coccidia by interfering with the passage of monovalent or divalent ions including sodium, potassium, calcium and magnesium, through the parasite's cell membrane (81, 102, 105). Additionally, in the market there are “mixed products” that are comprised of a combination of the two (10). Table 4 provides further characterization of these categories.

The extensive use of these anticoccidials prophylactically has resulted in a loss of efficacy of these compounds, triggered by increasing resistance by the parasite (11). To overcome this, anticoccidials are now used under programs called dual (or shuttle) or straight rotation. In the first program, two or more anticoccidials, usually with different modes of action, are alternated in the different feeds supplied during the chicken life cycle; whereas in the second program, the same drug is used continuously throughout a production cycle, but is changed for an alternative drug after one or several flocks (2, 8, 10).

Infection with Eimeria spp. triggers a variety of mechanisms in a host's immune system, resulting in an effective, long-lasting, but species-specific immunity (68). Generally, generating an immune response against Eimeria requires a large number of inoculating oocysts, with the exception of E. maxima, which is considered to be highly immunogenic and requires only a small number of oocysts to induce strong immunity (68). Additionally, the early endogenous stages of the parasite life cycle are considered to be more immunogenic than the later sexual stages (112). The immune system of birds is well-developed and when facing a challenge from an intestinal parasite like Eimeria (68), the gut-associated lymphoid tissue (GALT) is the first line of defense. This system has evolved into a specialized immune com-plex with organs such as Peyer's patches (PP), the bursa of Fabricius, and the cecal tonsils, hosting a variety of specialized immune cells such as epithelial, NK, and den-dritic cells (9). Infection stimulates a host response that begins with a non-specific immune response, mediated by macrophages, granulocytes, natural killer (NK) cells and soluble factors such as serum proteins. This precedes the development of an antigen-specific memory immune response mediated by lymphocytes (113) and their secretions, in the form of antibodies and cytokines (9, 68). The works of Rose et al. (114, 115) and Lillehoj and Choi (116) demonstrate the progression of the innate and adaptive responses to Eimeria infection in mouse and chicken models.

The first study that showed resistance in chickens against infection with E. tenella was reported by Beach and Corl in 1925; but it was not until 27 years later that the first live commercial vaccine against coccidiosis (CocciVac®) was launched in the United States (17, 62, 81, 105). Vaccination is included in coccidia control programs with the aim of inducing an immune response, generating protection against subsequent challenges with Eimeria spp., and decreasing the severity of coccidiosis (9, 10, 83).

Different vaccines are available on the market, including live virulent, live attenuated, and non-infectious derivatives of the parasite (subunit vaccines) (9, 117). Live vaccines use the oral introduction of low doses of Eimeria oocysts to stimulate humoral and cellular responses from the host immune system. The ingested oocysts generate a variety of antigens in different stages of the Eimeria life cycle to stimulate this response (8), with the cellular response being the most important in terms of resistance to the disease. This is supported by a scientific paper published by Lillehoj (118) in which birds were treated with Cyclosporin A to suppress cell-mediated immune responses, or treated with hormones to abrogate their humoral response by interfering with the bursa. The result was that birds with a hormonal bursectomy showed no alteration in their response to challenge with Eimeria tenella, while birds who had been previously treated with cyclosporin a showed increased susceptibility to the parasite.

These and other studies are crucial in the development of future vaccines. These studies are important to more fully understand the nature of the immune response against the parasite, as well as other interactions between the host and parasite (17, 62–65, 67–119).

Virulent strains isolated from the field without any alteration in their pathogenicity can be used as vaccines, as well (62), being highly effective and relatively cheap to produce. However, this practice also risks compromising flock performance and occurrence of clinical disease if managed incorrectly (120, 121). In mass vaccination, it is important to carefully standardize the dosage methods and conditions, enabling uniform inoculation within the flock, increasing oocyst shedding into the litter and reinfection of the flock. Animals that do not receive an appropriate dose of the vaccine may perpetuate subsequent infections that can cause asynchronous immunity, compromising the performance of the birds and increasing future susceptibility to the disease (9). These drawbacks have limited the use of virulent vaccine strains and they are not currently licensed for use in the European Union (122). However, this type the vaccine is used widely across much of North America, as well as in parts of Africa and Asia (121, 123).

The goal of attenuation is to decrease the pathogenicity of the parasite and therefore, its deleterious effects on the host. Several methods of attenuation have been used, including, selection for precociousness (124), irradiation (125), chemical treatment (126) and serial passage in chicken embryos (127). Precocious lines of Eimeria are characterized by a shortened endogenous life cycle due to the elimination of one or more schizogonies, leading to less damage to the intestinal tract and decreased production of oocysts. By selecting the first oocysts that are excreted in the feces to inoculate chickens during the second pass and repeating this process over and over, the reproductive potential and the pre-patent period of the Eimeria species selected is lowered. Along with these reductions there is a concomitant reduction in pathogenicity while the immunogenicity is retained (124). This type of vaccine is used extensively in much of Europe, as well as parts of Africa, Asia and Australasia, primarily with layer and breeder stock owing to their relative cost and limited productive capacity (123).

Identification of protective antigens is essential for the development of new vaccines against coccidiosis (128). Isolated and purified epitopes from virulent strains have been used in anticoccidial vaccines, mainly native or recombinant proteins expressed during various stages of development (sporogonies, merogonies, gametogonies) of the Eimeria parasite (62). This is particularly useful for maternal immunization, stimulating the production of large amounts of immunoglobulin Y (IgY), which is then transferred through the yolk of the egg, providing protective immunity to its offspring. This vaccination strategy can decrease the excretion of oocysts in birds challenged with Eimeria maxima up to 83%, and can provide some cross-protection against heterologous species like E. tenella and E. acervulina (128).

Attempts to develop next-generation recombinant anticoccidial vaccines have led to the identification of many potential vaccine antigens. Small-scale vaccination trials using antigens in recombinant protein, DNA or live-vectored formulations have been reported to achieve 30–90% reductions in parasite replication and/or gut lesion score, or comparable improvements in feed conversion ratio and/or body weight gain (122). However, one major constraint in deployment of such antigen-specific vaccines is an appropriate and effective delivery system (51). Several possible vectors for oral administration, including Bacillus, Salmonella, transgenic Eimeria and yeasts such as Saccharomyces cerevisiae, are currently in development and could be appropriate (51, 129, 130).

Currently, vaccination is a practice that is being promoted mainly due to demand for products satisfying a “No antibiotics, ever” label (51). In the past, it was a common practice only in breeder pullets and turkeys (131); this trend has been changing because public and legislative pressures are encouraging the search for cost-effective alternatives to anticoccidial drugs in broiler production, especially in countries such as the US, where (unlike the EU) ionophores are regulated as antibiotics (123). In response to these external pressures, 35–40% of US broiler companies have adopted annual cycles where two out of every six flocks receive anticoccidial vaccination instead of drugs (104). This practice is known as a bio-shuttle program in which vaccination of broilers on day of hatch is followed by the administration of grower and finisher diets containing anticoccidial drugs (132). This allows producers greater control in managing the risk of outbreak posed by the use of non-attenuated vaccines.

Another important advantage to using live vaccines for the control of coccidiosis is the replacement of Eimeria populations residing in the poultry house. This often has the effect of restoring the susceptibility of the Eimeria population in the house to traditional anticoccidial drugs, as evidenced in the work of Chapman and Jeffers (12). This study was an anticoccidial resistance trial in broiler chickens, tracking five consecutive flocks on a rotation schedule of anticoccidials. The use of vaccination in conjunction with salinomycin (ionophore) and diclazuril (chemical), restored sensitivity to these anticoccidials: they demonstrated that the anticoccidial programs that followed the vaccination program had improved sensitivity of the parasite to the anticoccidials.

Currently, difficulties including resistance to the cost of anticoccidials (133); consumer pressure for poultry products labeled as “antibiotic-free,” “no antibiotics ever,” or “raised without antibiotics”; (134), as well as the pathogenicity of live vaccines, is leading poultry producers worldwide to intensify their search for strategies that include safe, effective and economically viable alternatives for controlling coccidiosis (11, 133).

These alternatives include prebiotics, probiotics, essential oils, organic acids, antioxidants and nanobiotics (plant nanoparticles that have been used as antibacterial agents) (81, 135). Many of these compounds are used as dietary supplements with various applications including immune system stimulation, and anti-inflammatory and antioxidant action (2, 133). For example, the work Ali et al. (69), showed the anticoccidial effect of garlic (Allium sativum) and ginger (Zingiber officinale) against experimentally induced coccidiosis. Feed intake, body weight and feed conversion ratio (FCR) were significantly improved in ginger and garlic supplemented birds compared to the positive control (infected without additives). Similarly, oocyst shedding, lesion score and histopathology of the small intestines improved in ginger and garlic supplemented birds after challenge.

Table 5 shows some natural compounds that have been used for Eimeria control, their mechanism of action, and their efficacy in controlling coccidiosis (140). Mixed results in many studies show the need for further research into the potential of these alternative control strategies. For example, Scheurer et al. (137) examined three phytogenic compounds (oregano; combination of Curcuma, saponins, and inulin; Quillaja), and showed that there were no effects against Coccidiosis. Similarly, Idris et al. (141) reported that the use of essential oils as an alternative to anticoccidials is limited because of their antinutritional factors, toxicity, low dose effectiveness and their reduced protective response.

Further investigations should explore their mechanisms of action, and their protective response should be evaluated alone or in combination with a vaccine (141), as future control strategies are likely to include combinations of products as replacements for traditional anticoccidials.