To My Quyen, Nguyen Khanh Thuan, Nguyen Phuc Khanh, Nguyen Thanh Lam*

1. Introduction 

Coccidiosis is caused by protozoa of the phylum Apicomplexa, family Eimeriidae. In poultry, most species belong to the genus Eimeria and infect various sites in the intestine. The infectious process is rapid (4–7 days) and is characterized by parasite replication in host cells with extensive damage to the intestinal mucosa. Poultry coccidia are generally host-specific, and the different species parasitize specific parts of the intestine. However, in game birds, including quail, the coccidia may parasitize the entire intestinal tract. coccidia are distributed worldwide in poultry, game birds reared in captivity, and wild birds (Richard W. Gerhold, 2022). 

2. Aetiology

2.1 Bacterial characteristics 

Coccidia are almost universally present in poultry-raising operations, but clinical disease occurs only after ingestion of relatively large numbers of sporulated oocysts by susceptible birds. Both clinically infected and recovered birds shed oocysts in their droppings, which contaminate feed, dust, water, litter, and soil. Oocysts may be transmitted by mechanical carriers (e.g., equipment, clothing, insects, farm workers, and other animals). Fresh oocysts are not infective until they sporulate; under optimal conditions (21°–32°C with adequate moisture and oxygen), this requires 1–2 days. The prepatent period is 4–7 days. Sporulated oocysts may survive for long periods, depending on environmental factors. Oocysts are resistant to some disinfectants commonly used around livestock but are killed by freezing or high environmental temperatures. 

Sporulated oocysts are subspherical, with a rough bilayered oocyst wall (0.8 μm thick). Oocysts measured 24.0 × 22.8 (20.4–26.4 × 18.3–25.9) μm, oocyst length/width (L/W) ratio, 1.10. Oocyst residuum, polar granule and the micropyle were absent. Sporocysts are elongate-ovoid, 11.0 × 7.3 (12.7–9.2 × 7.9–6.6) μm, sporocyst L/W ratio, 1.5 (1.3–1.7). A thin convex Stieda body and indistinct substieda bodies were present and the sporocyst residuum was composed of numerous small granules less than 1.0 μm in diameter dispersed randomly. Each sporocyst contained 2 sausage-shaped sporozoites in 

head-to-tail arrangement. The sporozoite nuclei were located centrally surrounded by refractile bodies (Yang et al., 2016).

2.2 Classification 

The National Center for Biotechnology Information (NCBI) divides the phylum Apicomplexa into two classes: Aconoidasida and Conoidasida (Figure 2). The class Aconoidasida is divided into two orders: Haemosporida and Piroplasmida (containing the genera Babesia and Theileria), while the class Conoidasida is divided into two subclasses: Coccidiasina (containing the genera Eimeria, Neospora and Sarcocystis, that belong to order Eucoccidiorida) and Gregarinasina (Figure 2). It is estimated that subclass Coccidiasina separated from the class Aconoidasida~705 million years ago (Martínez-Ocampo, 2018). 

2.3 Relationship between coccidiosis and other poultry diseases

The tissue damage and changes in intestinal tract permeability and function may allow colonization by various harmful bacteria, such as Clostridium perfringens, leading to necrotic enteritis or salmonellosis. Cecal coccidiosis (E. tenella) may contribute to increased severity of the blackhead organism (Histomonas meleagridis) in chickens. Experimental infections with the 2 organisms were characterized by a higher incidence of hepatic disease, as compared with monoinfection with Histomonas. Immunosuppressive diseases may act in concert with coccidiosis to produce a more severe disease. Marek’s disease may interfere with development of immunity to coccidiosis, and infectious bursal disease may exacerbate coccidiosis, placing a heavier burden on anticoccidial drugs (Swayne, 2013).

3. Epidemiology 

3.1 Susceptible hosts 

Host specificity is strict among the Eimeria infecting poultry. The chicken is the only natural host of the 7 species of Eimeria (E. acervulina, E. brunetti, E. maxima, E. mitis, E. necatrix, E. praecox, and E. tenella). Reports of these species of Eimeria infecting other birds can be considered spurious. Cross-transmission of Eimeria spp. from chickens to other host species has been unsuccessful except where severely immunocom promised birds were used (Swayne, 2013). 

Naïve chickens of all ages and breeds are susceptible to infection. However, immunity develops after mild infections, limiting further infection. Newly hatched chicks often have high levels of maternal antibodies, but it does not appear that this limits susceptibility. Outbreaks are common at 3-6 weeks of age. In special situations, infections may be seen as early as 1 week of age. Surveys of coccidia in broiler houses in Georgia demonstrated that oocysts of coccidia build up during the growth of a flock and then decline as the birds become immune to further infection. This self-limiting nature of coccidial infections is widely known in chickens and other poultry. There is no stimulation of cross-protective immunity between species of coccidia. Thus, more than 1 outbreak of coccidiosis is possible in the same flock, with different species involved in each. Breeder pullets and layer pullets are at greatest risk because they are kept on litter for 20 weeks or more. Normally, the infections with E. acer vulina, E. tenella, E. mitis, E. praecox, and E. maxima are seen at 3-6 weeks of age and then E. necatrix at 8-18 weeks of age. E. brunetti is seen both early and late. 

Coccidiosis rarely occurs in layers and breeders during the laying cycle because of prior exposure to coccidia and resulting immunity. If a flock is not exposed to a particular species early in life or if immunity is depressed because of other diseases, outbreaks may occur after layers are moved to production houses. Outbreaks of any species in layers can depress egg production (Swayne, 2013).

3.2 Transmissions and vectors

Ingestion of sporulated oocysts is the only natural method of transmission. Once infected, chickens may shed oocysts in the feces for several days or weeks. The oocysts in feces become infective through the process of sporulation in about 2 days. Susceptible birds in the same flock may ingest the oocysts through litter-pecking or the contamination of food or water. 

No natural intermediate hosts exist for the Eimeria spp., but oocysts can be spread mechanically by a multitude of vectors, including insects, contaminated equipment, wild birds, and dust. Oocysts generally are considered resistant to environmental extremes and to disinfectants, although survival time varies with conditions. Oocysts may survive for many weeks in soil, but survival in poultry litter is limited to a few days because of the heat and ammonia released by composting and the action of molds and bacteria. Viable oocysts have been reported from the dust inside and outside broiler houses, as well as from insects in poultry litter. The darkling beetle (Alphitobius diaperinus), common in broiler litter, is a mechanical carrier of oocysts. Transmission from one farm to another is facilitated by movement of personnel and equipment between farms and by the migration of wild birds, which may mechanically spread the oocysts. New farms may remain free of coccidia for most of the first grow-out of chickens until the introduction of coccidia to a completely susceptible flock. Such outbreaks, often more severe than those experienced on older farms, are often called the new house syndrome. 

Oocysts may survive for many weeks under optimal conditions but will be quickly killed by exposure to extreme temperatures or drying. Exposure to 55°C or freezing kills oocysts very quickly. Even 37°C kills oocysts when continued for 2-3 days. Sporozoites and sporocysts can be frozen in liquid nitrogen with an appropriate cryopreservation technique, but oocysts cannot be adequately infiltrated with cryoprotectorants to effect survival. The threat of coccidiosis is less during hot, dry weather and greater in cooler, damp weather (Swayne, 2013).

3.3 Environment factors 

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. Sanitization plays a major role in reducing the dissemination of the parasite, 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 (Mesa-Pineda et al., 2021).

4. Life cycle

The protozoans of the genus Eimeria have a direct life cycle, characterized by high tissue and host specificity, involving stages of asexual and sexual multiplication, with three development phases: the formation of schizogony (agamogony/merogony), gametogony (gamete formation for sexual reproduction), and sporogony. The sporozoites invade the enterocytes, changing into trophozoites and starting a parasitic feeding period that lasts ∼12–48h. The parasitophorous vacuole is formed, the trophozoite begins to enlarge, and the parasite nucleus performs multiple asexual divisions, forming the schizont or meront, which is full of merozoites. Approximately 3 days post infection, the mature schizont ruptures and releases the merozoites (Figure 3C), which are fusiform and have an apical complex that allows them to move and infect intestinal epithelial cells to form additional schizont generations that reproduce asexually. The number of phases of asexual reproduction is characteristic of each Eimeria species and is thought to be genetically programmed. The main purpose of this phase is to boost the number of merozoites within the host as preparation for the sexual reproduction phase, which is an important characteristic of every apicomplexan life cycle. When the asexual reproduction phase is complete, the sexual reproduction stage or gametogony begins, occurring in three events. The first is gametocytogenesis, in which gametocytes are produced from merozoites. Second, during gametogenesis, haploid micro and macrogametes are differentiated from the gametocytes. Finally, macrogametocytes are fertilized by microgametocytes (Figure 3D), producing diploid zygotes, at which point sexual reproduction is completed; meiosis proceeds, inside the protective oocyst wall, followed by mitosis to produce the infectious sporozoites. The micro and macrogametocytes are morphologically different. The macrogametocyte grows quickly and forms a single macrogamete, with polysaccharide granules and lipid droplets; while the microgametocyte matures, breaks up and releases many small biflagellate microgametes that area vehicle to deliver DNA. The amount of microgametes varies depending on the species, for example, E. acervulina can produce between 20 and 30, and E. maxima 100 or more. After fertilization, the oocyst is formed with an undifferentiated cytoplasmic mass which corresponds to the zygote; this mass is protected by a double wall of proteins and fats that give it great resistance to mechanical and chemical damage from the environment. The duration of the parasite’s endogenous or internal phase is determined by the time needed to complete asexual and sexual reproduction and form oocysts. Once the oocyst is excreted from the animal in the feces (Figure 3E), the third phase of the cycle, sporulation, takes places. If environmental conditions area adequate, the diploid oocyst initiates sporogony formation, which occurs in three stages (Mesa-Pineda et al., 2021).

5. Clinical signs, gross lesions and histopathology

5.1 Clinical signs and gross lesions

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. Infected birds present with ruffled feathers and signs of depression or drowsiness (Figure 4). Additionally, feed and water consumption are decreased, and the feces may be watery, whitish and occasionally bloody. This results in dehydration, impaired weight gain, and in the absence of treatment, death. Additionally, thermalabsorption is to reduced brush border enzyme activity and disruption of intestinal integrity (Mesa-Pineda et al., 2021).

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. Goblet cells represent an important defense mechanism in the intestinal tract, secreting glycoproteins of high molecular weight called mucins. Mucins are the first line of defense against intestinal pathogens and act to protect the epithelium from pathogens and irritants in the intestinal lumen. 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. However, this increase in mucin production can also be harmful, promoting secondary colonization by other pathogens such as Clostridium perfringens, Salmonella and certain viruses like Marek's disease virus or infectious bursal disease virus. This has the effect of further altering intestinal health by impairing metabolism and nutrient absorption.

For the evaluation of gross lesions, a standardized intestinal lesion scoring technique is used, 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. 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. Table 2 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. 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.
Table 2: Description of changes and score of intestinal lesions due to infection with Eimeria spp (Mesa-Pineda et al., 2021).

5.1.1 Eimeria maxima Tyzzer 1929 

Gross lesions and histopathology

Minimal tissue damage occurs with the asexual cycles, which develop superficially in the epithelial cells of the mucosa. When the sexual stages develop in deeper tissues on days 5–8 PI, lesions develop because of congestion and edema, cellular infiltration, and thickening of the mucosa. Foci of infection can be seen from the serosal surface because of microscopic hemorrhages. The intestine may be flaccid and filled with fluid, and the lumen often contains yellow or orange mucus and blood. This condition has been described as “ballooning.” Microscopic pathology is characterized by edema and cellular infiltration, developing schizonts through day 4, and sexual stages (macrogametes and microgametes) in deeper tissues on days 5–8. In severe infections, considerable disruption of the mucosa occurs.

5.1.2 Eimeria necatrix Johnson 1930 

Pathogenicity, gross lesions, and histopathology

E. necatrix along with E. tenella are the most pathogenic of the chicken coccidia. Infection with 104–105 sporulated oocysts is sufficient to cause severe weight loss, morbidity, and mortality. Survivors may be emaciated, suffer secondary infections, and lose pigmentation. Droppings of infected birds often contain blood, fluid, and mucus. Naturally occurring infections have caused mortality in excess of 25% in commercial flocks. In experimental infections, 100% mortality is possible. Layer pullets suffering outbreaks at 7–20 weeks of age may suffer mortality, morbidity, loss of uniformity, and decreased egg‐laying potential. Gross lesions may be seen as early as 2–3 days PI, associated with firstgeneration schizogony, but the severe lesions at 4–6 days PI are caused by second‐generation schizogony. The intestine may be ballooned, the mucosa thickened, and the lumen filled with fluid, blood, and tissue debris. From the serosal surface, the foci of infection can be seen as small white plaques or red petechiae. In dead birds, these lesions appear white and black, giving rise to the expression “salt and pepper” appearance. Smears examined microscopically on days 4–5 PI may contain numerous clusters of large (66 µm) schizonts, each containing hundreds of merozoites. Clusters of schizonts deep in the mucosa often penetrate the submucosa, damage the layers of smooth muscle, and destroy blood vessels. In these instances, the foci are large enough to be seen from the serosal surface. Later, scar tissue may be seen where epithelial regeneration is incomplete. Few pathogenic effects are seen with the invasion of the cecal mucosa by the third‐generation schizonts and gametocytes because of the nonclustering nature of these stages. The third‐generation schizonts produce only 6–16 merozoites. Lesions may extend from the ventriculus (gizzard) junction to the ileo‐cecal junction, causing dilation (ballooning) and thickening of the mucosa. The lumen may be filled with blood and pieces of mucosal tissue. Microscopic examination of smears from the mucosal surface reveals numerous clusters of large schizonts, which are characteristic for this species and distinguish it from others that overlap in habitat. Also, oocysts are found only in the ceca. Histopathology of midgut from affected birds reveals a submucosa and lamina propria crowded with large clusters of schizonts. Often, large areas of the mucosa are sloughed off, and the lesion may extend through the muscle layers to near the serosal membranes.

5.2 Histopathology

In the cecum, oocysts invaded the cecum mucosa and intestinal gland (Figure 7B), and serious karyopyknosis and necrocytosis were detected in cecum mucosa cells. In the ShiYingZi-PM group (Figure 7C), ShiYingZi-TH group (Figure 7E), and positive control groups (Figure 7D, monensin; Figure 7F, sulfachloropyrazine sodium), few oocysts were observed in the cecum mucosa cells, and the cecum mucosa cells were granular and exhibited vacuolar degeneration; in the uninfected-untreated group (Figure 7A), the cecum displayed a normal structure (Song et al., 2020).

In the liver, E. tenella infection induced serious hepatocyte necrosis and inflammatory cell infiltration (Figure 7H); granular and vacuolar degeneration appeared in the ShiYingZi-PM group (Figure 7I), ShiYingZi-TH group (Figure 7K), and positive control groups (Figure 7J, monensin; Figure 7L, sulfachloropyrazine sodium); without infection, the liver showed a normal structure (Figure 7G).

In the kidney, serious granular and vacuolar degeneration and cell necrosis were observed in the infected-untreated group (Figure 7N); mild granular and vacuolar degeneration were observed in the ShiYingZi-PM group (Figure 7O), ShiYingZi-TH group (Figure 7Q), and positive control groups (Figure 7P, monensin; Figure 7R, sulfachloropyrazine sodium); more severe inflammatory cell infiltration was observed in the sulfachloropyrazine sodium group (Figure 7R) than that in the ShiYingZi-TH group (Figure 7Q); and without infection, the kidney showed a normal structure (Figure 7I) (Song et al., 2020).

6. Diagnosis 

Coccidiosis can best be diagnosed from birds euthanized for immediate necropsy. The entire intestinal tract should be examined first from the serosal side and then from the mucosal side. A microscope should be available for viewing endogenous forms on questionable lesions. The finding of a few oocysts by microscopic examination of smears from the intestine indicates the presence of infection, but not a diagnosis of clinical coccidiosis. Coccidia and mild lesions are present in the intestines of birds 3–6 weeks old in most flocks despite the use of drugs to prevent coccidiosis. The gross lesions of the important species of coccidia are easily recognized by experienced diagnosticians. Questionable lesions should be examined by microscopy. Experimentally, the biological characteristics of Eimeria are adequate. Molecular diagnosis by PCR is often practiced when further confirmation is needed, or in surveys (see later). Under practical conditions, a diagnosis of coccidiosis is warranted when the gross lesions are serious. A diagnosis should be based on finding lesions and confirming microscopic stages on necropsy of representative birds from the flock, rather than from culls. Cryptosporidia may be found in chickens or turkeys but are easily differentiated because of their small size and location in the brush border of the mucosal cells (Swayne, 2013).

6.1 Microscopic examination 

Developing schizonts, gametocytes, and oocysts of coccidia may be seen in smears taken from the suspected lesion. A small amount of mucosal scraping should be diluted with saline on a slide and then covered with a coverslip. Oocysts or macrogametes are most easily seen, but in many cases, the lesion is caused by maturing schizonts. Diagnostic characteristics that are of value include the clusters of the large schizonts of E. necatrix and E. tenella, the small round oocysts of E. mitis, or the large oocysts and gametocytes of E. maxima. Presence of clusters of large schizonts in the midgut area is pathognomonic for E. necatrix, and a similar finding in the ceca indicates E. tenella. Oocysts associated with lesions in the duodenum include E. acervulina, or E. praecox, and oocysts associated with lesions in the lower gut are E. mitis or E. brunetti. Oocyst size and shape are not useful as diagnostic characteristics, because of the extensive overlapping in size of the species. However, the combination of oocyst size, location in the gut, and appearance of the lesions gives considerable confidence in diagnosis. Measurement of 20–30 oocysts of the predominant type of oocyst usually gives a good indication of the size of the unknown species. This information is useful in conjunction with other observations in the identification of species in field cases (Swayne, 2013).

6.2 Lesion scoring 

The severity of lesions is roughly proportional to the number of sporulated oocysts ingested by the bird and correlates with other parameters such as reduced weight gain, loss of skin pigmentation, and higher feed conversion. The most commonly used practice is based on the system devised by Johnson and Reid. In this technique, a score of 0–4 is assigned to a bird, where 0 = normal and 4 = the most severe lesion. This technique is most useful in experimental infections, where the dose of oocysts and medicaments are controlled, and the species are known. Even though the technique devised by Johnson and Reid was originally designed to score the severity of pure infections in a research setting, many veterinarians and parasitologists have adopted it to gauge the severity of natural infections in field work. Even though more than 1 species of coccidia may be present at some time, only 4 separate sections of the intestine are scored: (1) the duodenum (upper), with lesions of E. acervulina, (2) the midgut, from the duodenum past the yolk sac diverticulum, with lesions of E. maxima, E. praecox, E. necatrix, and E. mitis, (3) the lower small intestine from the yolk sac diverticulum to the cecal junctures, with lesions of E. mitis, E. necatrix, E. maxima, and E. brunetti, and (4) the ceca, where only E. tenella lesions are found. The appearance of lesions from different species varies greatly (Swayne, 2013).

6.3 Microscopic scoring 

As with lesion scores, the severity of coccidiosis can be judged by the number and appearance of parasite forms seen upon microscopic examination of smears from the mucosa, lumen, or feces. Microscopic scoring is particularly useful for detecting and rating species that do not produce easily seen gross lesions, such as E. mitis, and E. praecox. Diagnosticians using this technique often use a scale of 0–4 to indicate the number of oocysts seen/ unit area (Swayne, 2013).

6.4 Droppings scoring 

In laboratory infections, the droppings score may be used in the same manner as lesion score for a rapid and fairly reliable rating of the infection. The extent of abnormal droppings is rated on a scale of 0–4, where 4 = maximum diarrhea, with mucus, fluid, and/or blood. This technique has obvious complications where birds are infected with more than 1 species of Eimeria (Swayne, 2013).

6.5 Histopathology methods 

Ordinary methods in histopathology are satisfactory for routine examination of tissues infected with coccidia. Staining of sections with H&E or other common histologic stains will demonstrate developing stages and a microscopic lesion scoring method was developed. Specialized techniques will identify specific stages: staining with Schiff reagent gives a brilliant red color with the polysaccharide associated with the refractile body and with wall‐forming bodies in the macrogamete. Monoclonal antibodies conjugated with fluorescent markers such as fluorescein are very useful in research because specific stages of parts of cells can be readily identified (Swayne, 2013).

6.6 Molecular diagnosis of Coccidiosis 

Sequences of DNA unique to each Eimeria species are used to design oligonucleotide primers thus allowing selective amplification by PCR. Molecular‐based assays for detecting and differentiating Eimeria have been described that target genes such as the ribosomal RNA internal transcribed spacer regions 1 or 2 (ITS1, ITS2) or sequence characterized amplified regions (SCAR), the latter identified through a technique termed random amplified polymorphic DNA. Multiplex PCR techniques have been described that combine all primers for each Eimeria species in a single tube. Newer technology includes real‐time (quantitative) PCR and loop‐mediated isothermal amplification (LAMP) as an alternative to gel electrophoresis.

The DNA extraction method is one of the most critical steps, whether the parasite source is intestinal tissue, fecal droppings, or litter samples. Several DNA extraction methods have been reported. An oocyst rupturing step is needed, whether by bead‐beating or grinding, in a buffer containing EDTA (ethylenediaminetetraacetic acid) to prevent DNA degradation. The disrupted oocysts suspension is treated with phenol and chloroform followed by ethanol precipitation or use of commercial DNA extraction kits. The latter contain chaotropic agents to preserve DNA integrity and allow subsequent DNA purification by employing silica‐containing mini‐columns that selectively bind DNA. The objective in whatever DNA extraction method is chosen is to produce high quality DNA (Swayne, 2013).

6.7 Species composition in chicken fecal droppings 

The oocysts are concentrated by flotation in 1 M sucrose or saturated sodium chloride, then treated with 6.0% sodium hypochlorite (100% bleach) for 15 minutes at room temperature on an orbital rocker. The oocysts are then washed by suspension in deionized water and centrifugated at 1,4 g for 10 minutes at 4°C. This step is repeated 4–5 times to remove all residual bleach. The oocysts are then suspended in ASL buffer (Qiagen, Germantown, MD) and transferred to a bead‐beater tube containing about 200 mg 0.5 mm glass beads. The oocysts are disrupted on a bead‐beater (BioSpec Products, Inc., Bartlesville, OK) twice for 2 minutes with placement on wet ice between bead‐beatings. Eimeria oocyst DNA is extracted using the QIAamp Fast DNA Stool Mini‐Kit and then analyzed by ITS1 PCR using primers specific for E. acervulina, E. brunetti, E. maxima, E. mitis, E. necatrix, E. praecox, or E. tenella following described procedures. An internal standard is included in each amplification reaction. The Eimeria species in a sample are identified by the presence of a target band of predicted size. A laboratory has used this procedure for over 10 years to characterize the species composition of Eimeria in litter from commercial broiler houses. Depending on location, the most prevalent species in broiler houses in the United States appear to be E. acervulina and E. maxima, followed by E. praecox and E. tenella (Swayne, 2013).

7. Treatment

Many products are available for prevention or treatment of coccidiosis in chickens and turkeys. Detailed instructions for use are provided by all manufacturers to help users with management considerations and to ensure compliance with regulatory approvals.

Anticoccidials are given in the feed to prevent disease and the economic loss often associated with subacute infection. Prophylactic use is preferred, because most of the damage occurs before signs become apparent and because drugs cannot completely stop an outbreak. Therapeutic treatments are usually given by water because of the logistical restraints of feed administration. Antibiotics and increased levels of vitamins A and K are sometimes used in the ration to improve rate of recovery and prevent secondary infections (Richard W. Gerhold, 2022).

7.1 Characteristics of anticoccidial drugs

In spite of a higher demand for broiler chickens raised without antibiotics and/or drugs, fermentation‐derived ionophore and chemically synthetized anticoccidials remain the backbone of coccidiosis control. All types of drugs used for coccidiosis control are unique in their mode of action, the way in which parasites are killed or arrested, and the effects of the drug on the growth and performance of the bird. Following are the most important characteristics. 

Spectrum of activity

There are several important species of coccidia in chickens, several more in turkeys, and many others in other hosts. A drug may be efficacious against 1 or several of these parasites; very few drugs are equally efficacious against all (Swayne, 2013). 

Mode of action

Each class of chemical compound is unique in the type of action exerted on the parasite, and even in the developmental stage of the parasite most affected. The chemical mode of action of some drugs is known to be a highly detailed event, and the action of other drugs remains a mystery. The sulfonamides and related drugs compete for the incorporation of paraaminobenzoic acid and metabolism of folic acid. Amprolium competes for absorption of thiamine by the parasite. The quinoline coccidiostats like clopidol inhibit energy metabolism in the cytochrome system of the coccidia. The polyether ionophores upset the osmotic balance of the protozoan cell by altering the permeability of cell membranes for alkaline metal cations (Swayne, 2013). 

Endogenous stage affected

The coccidia are prone to attack by drugs at various stages in the development in the host. Totally unrelated drugs may attack the same stage of parasite. The quinolones and ionophores arrest or kill the sporozoite or early trophozoite. Nicarbazin, robenidine, and zoalene destroy the first‐ or secondgeneration schizonts, and the sulfonamides act on the developing schizonts and on the sexual stages. Diclazuril acts in early schizogony with E. tenella but is delayed to later schizogony with E. acervulina and to the maturing macrogamete with E. maxima. The time of action in the life cycle has been construed as having significance in the use of drugs in certain types of programs in which immunity is desired, but there is no good evidence that this is the case under field conditions. 

Coccidiocidal vs. Coccidiostatic medications

Some drugs kill the parasite, but others only arrest development. When coccidiostatic medication is withdrawn, arrested parasites may continue to develop and contaminate the environment with oocysts. In such cases, a relapse of coccidiosis is possible. In general, the coccidiocidal drugs have been more effective than those that are coccidiostatic (Swayne, 2013). 

Effects of drugs on the target animal

Most compounds used in animal feeds have good selective toxicity, providing toxicity for the parasite but being nontoxic to vertebrates. Unfortunately, toxicity and side effects of drugs on the host are possible where formulation errors lead to overdose. Sometimes, a drug may exhibit side effects at the recommended use level. Some of the toxicity may be the result of management, genetics, nutrition, or other interaction, and in other cases, the margin of safety is just too narrow. Environmental interaction is possible with nicarbazin, which interacts with high temperatures and high humidity to produce excess mortality. Nicarbazin also has adverse effects in layers, causing a bleaching of brown‐shelled eggs, mottling of yolks, reduced hatchability, and reduced egg production. The ionophores are highly toxic at elevated doses, causing a transient paralysis in mild overdoses or a permanent paralysis and mortality in more severe cases. Monensin was once thought to interact with methionine to reduce feather growth, but this relationship is not clear. Under some conditions, lasalocid will stimulate water consumption and excretion, resulting in a wet litter. With slight overdoses, most of the ionophores depress weight gain under laboratory conditions. A withdrawal period of 5–7 days is often practiced to allow compensatory growth to make up for the lost gain. The ionophores are known for their toxicity to other animals. For example, monensin and salinomycin are highly toxic to horses. The LD50 for monensin in horses is about 2 mg/kg body weight. Salinomycin and narasin are highly toxic to turkeys and cause excessive mortality at the levels recommended for use in chickens, whereas monensin and lasalocid are well tolerated in turkeys at the level used for chickens (Swayne, 2013).

Programs for use of anticoccidial drugs in broilers 

The objective in broilers is to produce the maximum growth and feed efficiency with minimum of disease. In long‐lived birds like table‐egg layers and breeders kept on the floor, the objective is to protect against early acute infections and to provide long‐lasting immunity. The choice of a product or program may depend on the season of the year or other factors which affect exposure. The following several types of programs are practiced (Swayne, 2013). 

Continuous use of a single drug

Often, a single product will be used from day 1 to slaughter, or with a withdrawal period of 3–7 days. Most products are approved for use until slaughter, but producers withdraw medication for economic or other reasons, such as the compensatory gain previously mentioned when ionophores are withdrawn from the feed (Swayne, 2013).

Shuttle or dual programs

The use of 1 product in the starter feed and another in the grower feed is called a shuttle program in the United States and a dual program in other countries. Some programs might contain as many as 3 drugs, with 1 drug in the starter, another in the grower, and yet another in the finisher. The shuttle program usually is intended to improve coccidiosis control. Intensive use of the polyether ionophore drugs for many years has produced strains of coccidia in the field that have reduced sensitivity to them. It is a common practice to use another drug such as nicarbazin, diclazuril, or clopidol in either the starter or grower feed to bolster the anticoccidial control and take some pressure off of the ionophore. In other cases, the order of these drugs is reversed. The use of shuttle programs is thought to reduce buildup of drug resistance. Historically, a high percentage of producers use some type of shuttle program (Swayne, 2013). 

Rotation of products

It is considered sound management to make periodic changes in anticoccidial drug use. Most producers in the United States consider changes in the spring and fall. Rotation of drugs may improve productivity because of the build‐up of isolates or species of coccidia that have reduced sensitivity after products have been used for a long time. Producers often notice a boost in productivity for a few months after a change of anticoccidial drugs. A similar effect has been demonstrated when live coccidiosis vaccines are used because all vaccines contain strains of Eimeria species susceptible to all the anticoccidial drugs currently on the market. The seasonal rotation of products is intended to correspond with the intrinsic properties of the drugs. In the United States, nicarbazin is used principally in the cooler months of the year, which also corresponds with maximum coccidiosis challenge. In the summer months, coccidiosis challenge tends to be milder, so weaker anticoccidials or live coccidiosis vaccines are preferred (Swayne, 2013).

Drug resistance 

The development of tolerance to drugs by coccidia after exposure to medication is the most serious limitation to the effectiveness of these products. Surveys reveal widespread drug resistance in coccidia in the United States, South America, and Europe. Even though coccidia develop less resistance to some drugs than others, long‐term exposure to any drug will produce a loss in sensitivity and, eventually, resistance. Drug resistance is a genetic phenomenon, and when established in a line of coccidia, will remain for many years, or until selection pressure and genetic drift force return to sensitivity in the population. Drugs such as the quinolones like clopidol have a well‐defined mode of action, and resistance develops quickly as coccidia are selected with cytochromes, which do not bind as readily to the drug. As an example, the emergence of resistance to decoquinate has been studied and documented in commercial broilers. The polyether ionophores, in contrast, have a more complicated mode of action involving the mechanisms of active transport of alkaline metal cations across cell membranes, and it has taken many years for coccidia to become tolerant, and in some cases, completely resistant. Many other drugs appear to be intermediate in selecting resistance in coccidia. The primary defense against drug resistance is the use of less intensive programs, shuttle programs, and frequent rotation of drugs and vaccines. Rotation of programs, used alone, will not prevent the development of resistance. In some instances, coccidia are able to become resistant to drugs after only a few months of use, and once developed, drug resistance is slow to dissipate. In recent years it has become a common practice to incorporate live coccidiosis vaccines in the rotation program, reasoning that the drug sensitive vaccine strains tend to replace the drug resistant wild types. This approach has had demonstrable effects on the drug sensitivity profile on farms where it has been practiced (Swayne, 2013).

Anticoccidial drugs 

Some products are still available commercially, but the approvals remain. Those used at present include monensin, narasin, salinomycin, semduramicin, lasalocid (polyether ionophores), diclazuril, nicarbazin, amprolium, decoquinate, clopidol, sulfadimethoxine/ormetoprim, and sulfaquinoxaline. A product combining narasin with nicarbazin is also used to take advantage of synergism between these molecules. Other products listed with approvals but lacking in significant activity include chlortetracycline and oxytetracycline. These products may prevent mortality from coccidiosis when given at high levels because of antibacterial activity but are not of much value in general use. The polyether ionophores became the drugs of choice for prevention of coccidiosis in 1972 and remain the most extensively used today. Other drugs, such as clopidol, diclazuril, halofuginone, nicarbazin, and robenidine, are used mostly in shuttle programs as an adjunct to the ionophores. In spite of the challenges posed by drug resistance anticoccidial drugs still remain the primary means of coccidiosis control worldwide. Unlike the European Union, in the United States ionophore anticoccidials are classified as antibiotics and therefore cannot be used in poultry sold with any claims to having been raised without antibiotics, raised without antibiotics (RWA), no‐antibiotics ever (NAE), organic, etc. This creates a serious problem for the long‐term prevention and control of coccidiosis in poultry because producers must rely exclusively on chemically synthetized anticoccidials and live coccidiosis vaccines (Swayne, 2013). 

Other diseases are made worse as a result, particularly necrotic enteritis. Chemically synthetized anticoccidials have no anticlostridial activity, and it is well known that live infection (even vaccination) with coccidian exacerbates clostridial infections (Swayne, 2013). 

For the treatment and control of coccidiosis in poultry, VICOX Toltra-oral solution is highly effective against the following species of Coccidia: Eimeria acervulina, E. adenoides, E. brunetti, E. maxima, E. tenella, E. meleagrimitis and E. necatrix. Additionally, VICOX toltra doesn't affect the rate of laying, on fertility and growth rate.

Dosage

For poultry only.     

1 m/liter of drinking water (25 ppm) for 2 consecutive days. 

Medicated water should be the only source of drinking water.

8. Control and prevention 

8.1 Immunization

Medication Programs in Broilers Chickens develop immunity to coccidiosis after natural exposure and may even develop substantial immunity while receiving anticoccidial drugs. The poultry industry has learned to take advantage of this phenomenon, practicing longer withdrawal programs of 2–3 weeks or even longer in some instances (Swayne, 2013).

8.2 Coccidiosis vaccines 

Considerable research on coccidiosis vaccines in recent years has produced new live products. Table 3 lists the live coccidiosis vaccines currently approved for sale in broilers, breeders, and layers in the United States. Increasingly, these products are finding use in the broiler industry. When live sporulated oocysts of coccidia are given to chickens at an early age, immunity against the species contained in the inoculum is stimulated. The pathogenicity of coccidia in these vaccines is attenuated largely by the size of the dose and by the means of administration. Some vaccines sold in the United States or internationally contain modified live coccidian, attenuated by genetic selection for short life cycle development (precociousness). The use of coccidiosis vaccines in broilers has been limited by the possibility of adverse reactions, particularly a negative effect on feed efficiency. More recent advances in administration methods have overcome much of this limitation. The Coccivac products pioneered in this growing family now includes several other live vaccines produced by various manufacturers in many countries (Coccivac, Immucox, Hatchpack Cocci‐III, Paracox, Livacox, Bio‐Coccivet, Advent, In‐Ovo Cox, Eimeriavax, Evalon, and others). Some live vaccines have been prepared from attenuated lines of oocysts (e.g., Hatch‐pack Cocci‐III, Paracox 7, and Livacox 7). These vaccines normally contain 3 or more species of Eimeria, which are thought to be the most important. The Eimeria infecting poultry immunize only against themselves, so that the vaccine will only protect against the included species. In the case of broiler vaccines, these are E. acervulina, E. maxima, and E. tenella. The success of some vaccines may depend more on a novel administration technique rather than attenuation. One experimental product was encapsulated in alginate beads and then mixed into the starter feed for “trickle administration”. Other methods presently used are spray cabinet administration, direct eye‐spray, in ovo inoculation, or spraying the oocysts directly into feed or mixing in water in the poultry house. One product was mixed into gels, which were placed into the chick boxes for the chicks to eat. Other experimental approaches include inoculation of parasites or antigens in ovo and inoculation via the yolk sac diverticulum (Swayne, 2013). 

Several reviews have been published that deal with the use of vaccines for the prevention of coccidiosis in poultry. 

Table 3: Live coccidiosis vaccines approved for sale in the United States (Swayne, 2013)

All vaccines for broilers contain at least Eimeria acervulina, E. maxima and E. tenella and all vaccines for breeders contain at least E. acervulina, E. brunetti, E. maxima, E. necatrix and E. tenella. ² Currently not produced by the manufacturer.

In the United States, administration of live coccidiosis vaccines worked well, particularly during the summer months when the arsenical compound, roxarsone, was available. Following the voluntary halt in sales of roxarsone by the manufacturer (at that time Pfizer, Inc.) over concerns of conversion of organic arsenic to inorganic arsenic (a recognized carcinogen) live coccidiosis vaccines struggled. Roxarsone is classified by the US FDA (Food and Drug Administration) as a nonantibiotic growth promoter with some anticoccidial activity against E. tenella. McDougald et. al. demonstrated that roxarsone had good anticoccidial activity against E. tenella and that the performance improvements seen when roxarsone is administered to broiler chickens in combination with anticoccidals came primarily from its anticoccidial properties. Other research has demonstrated that roxarsone also has significant anticlostridial activity. In order to remedy the performance issues encountered following the halt in sales of roxarsone, vaccine manufacturers and broiler producers have resorted to what it is now known as a hybrid program or a bio‐shuttle program. In this program, an anticoccidial drug (an ionophore or a chemically synthetized anticoccidial in non‐NAE production or a chemically synthetized anticoccidal in NAE production) is added to the feed at a low concentration for a specified period of time to reduce oocyst shedding and prevent adverse effects on performance. In order to prevent interference with immunity development the anticoccidial is generally added to the grower feed starting at 16–18 days of age. 

Another approach to coccidiosis control includes the use of coccidian proteins, which have a protective effect when administered to chicks. These proteins can be made in quantity if the gene that encodes the protein is cloned into a bacterial cell. Research identified broadspectrum antigens and appropriate routes of administration. One product based on this approach is CoxAbic, which is composed of an antigen developed from a monoclonal protein produced in the gametocyte of E. maxima. CoxAbic is given to hens in 2 doses to confer maternal protection during the first 3 weeks of brooding (Swayne, 2013).

8.3 Control programs used in breeders and layers 

Pullets started on the floor and later reared as caged layers are not as dependent on immunity to coccidiosis as are floor layers. Like broilers, they are often protected against coccidiosis with preventive medication, until they are moved to cages. Breeder pullets that will be kept on the floor during lay should have immunity to coccidiosis and may be vaccinated. Controlled exposure vaccination can be given by means of commercially produced live products (described above). Natural or accidental exposure assumes the presence of oocysts of important species. A broad‐spectrum anticoccidial drug is sometimes given at the lowest approved level to provide protection for 6–12 weeks. Some producers reduce the level of the drug during the final 4 weeks in a step‐down program, although as mentioned previously, chickens tend to develop immunizing infections despite the presence of the drug. This approach is aimed at allowing moderate numbers of coccidia to develop in the birds, stimulating the host immune system to protect against serious outbreaks. Such exposure usually is sufficient to protect against all species. Outbreaks of E. necatrix have sometimes occurred at 8–16 weeks, after all medication has been stopped. Climatic and seasonal conditions may add to the inherent uncertainties of this method (Swayne, 2013).

Disinfection and sanitation

Older recommendations for coccidiosis control often suggest directions for sanitation and disinfection to prevent outbreaks. Most of these are no longer considered valid because: (1) there have been too many failures in such programs; (2) oocysts are extremely resistant to common disinfectants; (3) complete house sterilization is never complete; and (4) an oocyst‐sterile environment for floor‐maintained birds could prevent early establishment of immunity and allow late outbreaks. In addition to disinfectants normally used in poultry houses, specific products have been used to target the oocyst for destruction. Chickens reared in banks of cages often suffer outbreaks of coccidiosis. The concentration of susceptible birds in stacked laying cage batteries, and the presence of mechanical vectors, such as flies, make birds particularly vulnerable to infection (Swayne, 2013).

9. References 

Martínez-Ocampo, F., 2018. Genomics of Apicomplexa, Farm Animals Diseases, Recent Omic Trends and New Strategies of Treatment. IntechOpen.

Mesa-Pineda, C., Navarro-Ruíz, J.L., López-Osorio, S., Chaparro-Gutiérrez, J.J., Gómez-Osorio, L.M., 2021. Chicken Coccidiosis: From the Parasite Lifecycle to Control of the Disease. Frontiers in Veterinary Science 8.

Richard W. Gerhold, J., 2022. Overview of Coccidiosis in Poultry. MSD Manual. Veterinary Manual.

Song, X., Li, Y., Chen, S., Jia, R., Huang, Y., Zou, Y., Li, L., Zhao, X., Yin, Z., 2020. Anticoccidial Effect of Herbal Powder “Shi Ying Zi” in Chickens Infected with Eimeria tenella. Animals 10, 1484.

Swayne, D.E., 2013. Diseases of Poultry. John Wiley & Sons.

Yang, R., Brice, B., Ryan, U., 2016. Morphological and molecular characterization of Eimeria purpureicephali n. sp. (Apicomplexa: Eimeriidae) in a red-capped parrot (Purpureicephalus spurius, Kuhl, 1820) in Western Australia. International Journal for Parasitology: Parasites and Wildlife 5, 34-39.