Control Tools

• Diagnostics availability

• Commercial diagnostic kits available worldwide

  Species differentiation methods classically rely on biological features including: oocyst morphology (shape, size, micropyle, oocyst residuum) prepatent period, minimum sporulation time, intestinal site of colonization, characterisation of pathological lesions seen post-mortem and/or viewed histologically. In the hands of experienced operators these are reliable and informative techniques but not amenable for high throughput.

  PCR assays are available for the seven well-established species of Eimeria that infect the chicken, mainly targeted to the ITS rRNA sequences and Sequence-Characterized Amplified Region (SCAR) markers. These seven Eimeria species can be simultaneously detected and discriminated in a single-tube multiplex PCR assay. Quantitative PCR assays are also developed for these seven species.

  PCR and quantitative PCR assays are also available for turkey Eimeria species.

  PCR methods are used in specialised laboratories, for example companies who manufacture vaccines and evaluate performance in the field. Automated methods using microscopy and AI-based software for counting and speciation will be available soon for the EU market.

  List of commercially available diagnostics kits (Diagnostics for Animals).

  GAPS :

  Validated PCR and quantitative PCR assays for newly described species of chicken Eimeria (formerly described as OTUs) are not published.

  There are no rapid/ex vivo tests to differentiate drug sensitive from drug resistant parasites.

  There are no rapid/ex vivo tests to differentiate attenuated vaccine strains from wild-type parasites.

  Automated imaging methods may have some limitations but could provide faster results than the PCR-based assays.

  None of the rapid assays give information on the intensity of pathology (damage to the intestinal tract) caused by parasite infection.

• Diagnostic kits validated by International, European or National Standards

  No diagnostic method has been validated; there are no National, European or International Standards.

• Diagnostic method(s) described by International, European or National standards

  No diagnostic method has been adopted officially; classical lesion scoring by an experience operator remains the gold standard for assessing the severity of infection in chickens.

• Commercial potential for diagnostic kits in Europe

  Hard to judge if there would be a mass market for this with current technology as tests are relatively expensive. There is a market in specialised laboratories for flock monitoring.

  GAPS :

  Simple testing that could be carried out by veterinarians or technicians (eg lateral flow tests) could be useful but there are technical issues to overcome as test materials are faeces containing tough oocysts, or gut scrapings containing endogenous stages.

  Furthermore, the simple act of identifying the presence of some coccidial species may not be linked with disease or impact on gut health, or growth, so must be interpreted with caution.

• DIVA tests required and/or available

  No DIVA tests are available, and no assays are published. Some vaccine manufacturers may have this capability in-house.

  GAPS :

  DIVA tests that differentiate live vaccine strains from field strains would be useful to determine efficacy of live vaccines in replacing pathogenic/drug-resistant field strains. They would also be useful to evaluate or dismiss the possibility that vaccine strains are causing disease.

• Vaccines availability

• Commercial vaccines availability (globally)

  Live vaccines are available against seven well-established species of Eimeria that infect chickens. Each vaccine comprises specific numbers of sporulated oocysts of different species /strains depending on the target market (i.e. broilers or breeders/ layers).

  Live oocyst vaccines are applied by various routes, according to national or regional regulations including hatchery spray, spray on birds, spray on feed, in drinking water, in eye-drops, in gel application, or in ovo administration.

  Non-attenuated live coccidiosis vaccines contain sporulated oocysts not modified or selected for reduced virulence; some contain field isolates regarded as less pathogenic than classically studied strains. Non-attenuated vaccines must be delivered carefully to ensure all chicks receive the lowest dose that induces robust immunity against subsequent challenge (including that arising from recycling of the vaccine) without causing serious damage to the intestinal tract.

  There are many non-attenuated live coccidiosis vaccines available around the world, mostly produced and marketed locally. None are approved for use in Europe.

  Attenuated live coccidiosis vaccines contain sporulated oocysts from stable parasite strains or populations that are deliberately selected for reduced virulence. They have a much higher margin of safety than non-attenuated vaccines. In almost all cases, attenuated parasites are selected by passaging parasites through chickens and collecting the earliest emerging oocysts. After many passages (variable according to parasite species) desirable and genetically stable precocious phenotypes are obtained in which parasites have shorter pre-patent times, reduced virulence and growth (due to loss or reduction of replicative stages) but retain full immunogenicity.

  MSD Animal Health, Huvepharma and Hipra each produce attenuated live vaccines for layer/breeders and broiler chickens with market authorization throughout Europe.

  Biopharm produces broiler and breeder/layer vaccines for chickens that are approved in some markets, including some but not all countries of Europe. In these vaccines, the Eimeria tenella component is not precocious but was attenuated by adaptation to growth in embryonated hens’ eggs.

  A sub-unit vaccine from Phibro formulated as an oil emulsion for maternal immunisation of broiler breeders to provide passive protection to broiler chicks was licensed in selected countries but is no longer available.

  For turkeys, one virulent live vaccine containing selected species of turkey Eimeria oocysts for application in water, by gel administration or by hatchery spray is approved in some countries but not in Europe. Hence, there are no anticoccidial vaccines for turkeys currently available in Europe.

  GAPS :

  Non-attenuated vaccines are cheap to produce because they contain wild-type parasites that grow to high numbers in chickens and each vaccine dose requires small numbers of oocysts from each parasite component.

  Attenuated (precocious) lines or populations have significantly reduced replication compared to non-attenuated parasites. This makes them much safer but large numbers of chickens are needed to grow them which drives up overall costs and can limit production of some components.

  In Europe, all coccidiosis vaccines must be produced in SPF chickens so improved industry coordination (between SPF producers and vaccine producers) would be beneficial.

  With a short shelf-life, unexpected gaps or breakdowns in commercial production cycles pose risks to vaccine supply chains.

  There is a need for an EU licensed anticoccidial vaccine for turkeys and for all other minor species (eg pheasant, partridge, guinea fowl, quail, duck, goose); this is especially urgent for those reared without anticoccidial feed additives.

• Marker vaccines available worldwide

  None. Live attenuated vaccine lines possess traits that have the potential to differentiate them from wild type lines. It is however not currently practical to do this on a routine basis.

  GAPS :

  Genetic characterization of live attenuated lines will allow the development of molecular methods for the differentiation of attenuated versus wild type field isolates.

• Effectiveness of vaccines / Main shortcomings of current vaccines

  Current live vaccines (virulent or attenuated) are efficient in stimulating protective immunity. However, these vaccines rely on recycling of oocysts via faeces in the litter to build up robust immunity so are susceptible to problems when environmental conditions are not conducive to oocyst survival, or where birds have limited contact with faeces, for example in aviaries for pullets, or when caged.

  If not administered carefully, live non-attenuated vaccines can be associated with direct effects on production or predispose birds to other diseases. Non-attenuated vaccines also may introduce species that are generally rare but highly pathogenic in the field, such as Eimeria brunetti or Eimeria necatrix. These can pose a risk to subsequent flocks, increasing environmental loads and potentially causing disease down the line.

  Immunity induced by live vaccines is highly species-specific so vaccines must include oocysts from all the species for which protection is being claimed.

  GAPS :

  Live coccidiosis vaccines do not induce cross-protection against species not included in the vaccine. In some cases, they do not induce robust protection against all variant strains of a species.

  All current vaccines must be produced in chickens, for Europe this is SPF chickens, which is costly.

  With most current vaccines, full immune protection is acquired after ~three weeks and relies on boosting from recycled parasites. If there is a high environmental load of parasites (eg contaminated litter), young birds will remain susceptible to disease. One vaccine (Hipra Evant) is administered with a Montanide-based adjuvant and has an onset of immunity of 2 weeks.

  It would be beneficial to develop vaccines that have reduced dependency on oocyst recycling along with methods to vaccinate in conditions where birds do not have access to faeces.

  Vaccines are needed that have longer shelf life with no cold chain.

• Commercial potential for vaccines in Europe

  With continued rapid growth of the global broiler market there is significant commercial potential for additional and novel anticoccidial vaccines.

  In the USA there is pressure from retailers and consumers to market chicken products as having ‘No Antibiotics Ever’ status. This is pushing the balance in favour of vaccination (mostly with non-attenuated vaccines) and against prophylactic anticoccidials, which are categorised in the USA as antimicrobials, rather than feed additives.

  In Europe where in-feed anticoccidials are regarded as a crucial tool for raising conventionally reared broilers, the licenced vaccines (attenuated only) have a relatively small market share. This is reflected in recent position papers from the Federation of Veterinarians of Europe (FVE) and the Poultry Veterinary Study Group (PSVG) of the EU.

  In Europe, vaccination of breeders with live-attenuated coccidiosis vaccines is common practice whilst the percentage vaccination of layers varies between countries and there is potential for growth in this segment. Vaccines are also often used in elite and organic production of broilers. For conventional broilers, vaccination is becoming more popular in Spain and Italy. In some countries some producers incorporate vaccines into rotational control programmes (where drugs and vaccines are used in successive flocks). Nevertheless, overall in Europe there is a significant potential market for vaccines for conventional broilers. Field data collected by companies already making and marketing vaccines indicate similar performances of flocks treated with vaccines or anticoccidials.

  Several research groups globally have identified candidate antigens from major species (mainly E. tenella but some work on E. maxima and E. acervulina) that show reasonable to good efficacy in small-scale experimental trials. These antigens could form the basis of subunit or recombinant vaccines. A variety of delivery methods (DNA, adjuvanted recombinant protein, yeast-vectored, viral-vectored, transgenic Eimeria) have been trialled. The potential to move these kinds of products forward will rely on strong partnerships between academic researchers, the veterinary vaccine industry and poultry producers.

  GAPS :

  There is potential to improve the manufacturing process of current vaccines if reliable in vitro methods for production of oocysts could be developed.

  There is a need for rigorous evaluation of the impact of current vaccines on broiler performance and gut health.

  In developing new vaccines, the priority is to develop cost-effective vaccines suitable for mass application; this is critical within the global perspective where cost, ease of use, shelf-life and absence of cold-chain are critical components.

  Ideally, novel vaccines would be multivalent and induce cross-protection against more than one parasite species.

  Novel vaccines eg those based on subunit or recombinant technology should induce good cellular immunity as this is a key component of the protective response to coccidiosis.

  There is a need for closer collaboration between academic researchers, commercial vaccine producers, and the poultry sector (breeders and producers) to develop stronger links and pipelines that can support translation of research findings.

• Regulatory and/or policy challenges to approval

  There are no likely challenges to developing and registering additional live oocyst-based vaccines.

  If a subunit or recombinant vaccine were to be taken forward there should be no insurmountable challenges; in the event of a transgenic parasite vaccine being preferred, this would be a novel approach that would require expert evaluation.

  GAPS :

  The draft monograph on live coccidiosis vaccines stipulates the use of live birds for evaluation of potency. Alternative methods should be pursued to replace the use of live birds.

  There is a need to prepare a monograph on future subunit vaccines to outline what would be important in the case of registration.

  For turkeys, there is no easy access to SPF flocks and in the USA, production of vaccines for both chickens and turkeys is done with ‘healthy’ flocks. This is something that should be investigated also in Europe.

• Commercial feasibility (e.g manufacturing)

  For as long as use of anticoccidial drugs remains possible, vaccines for broilers will only be competitive if costs of manufacture are kept low and there is minimal risk of them having negative impacts on bird performance.

  GAPS :

  Any adoption of an in vitro culture system for commercial production of live oocyst-based vaccines would require a dedicated high-throughput culture system.

  There is a gap in judging the cost/benefit balance of vaccines compared to anticoccidial feed additives because of the impacts of subclinical infections which are not recognised/evaluated.

• Opportunity for barrier protection

  Eimeria are ubiquitous in the environment and in consequence barrier protection is not considered a feasible option for complete control. However, thorough cleaning and disinfection with an effective oocysticide does provide some barrier protection, reducing environmental challenge in the early phase of a new grow out.

  GAPS :

  Much evaluation of oocysticides is based on tests with Cryptosporidium parvum, which has a very different oocyst structure to that of the Eimeria species. Dedicated tests for efficacy against Eimeria that take into account the conditions in poultry buildings would be useful.

• Opportunity for new developments

  A number of research groups globally are actively involved in the identification of protective antigens. If suitable candidates providing acceptable levels of efficacy, can be found, delivery methods capable of overcoming the shortcomings identified above may be developed within a 5 to 10 year timescale.

  There is potential to improve the manufacturing process should a reliable in vitro method for the production of oocysts be developed.

  GAP: New delivery methods for vaccines.

• Pharmaceutical availability

• Current therapy (curative and preventive)

  Anticoccidial drugs (ionophores and synthetic compounds) are available and widely used as additives in the feed for the prevention of coccidiosis. Although resistance by the parasites is described for all marketed products, most poultry producers in Europe are using combination products or ionophores for prevention of coccidiosis in broilers.

  There is a trend for registration of combination products (ionophore + synthetic compounds) as they prove to be safer than the individual compounds (lower concentrations of synthetics) and efficacious.

  A few anticoccidial drugs are available to treat birds suffering from coccidiosis usually by including a soluble compound in the drinking water. The results can be equivocal, mainly due to resistance acquired against these synthetic molecules.

  Drug withdrawal periods vary from 0 to 5 days before slaughter. Eight of the thirteen registered anticoccidial feed additives for broilers in the EU have a withdrawal period of 0 days.

  Several products consisting of mixtures of plant extracts and/or essential oils are widely used in the field, with no official claim and no registration concerning anticoccidial activity. No relevant feedback is available on their efficacy and usefulness.

  GAPS :

  There is a need for new anticoccidial drugs with slow resistance development and research to generate better knowledge on drug mechanisms and how resistance develops.

  Better therapeutic drugs are needed to treat outbreaks of clinical disease and with shorter withdrawal periods (for example, toltrazuril is not useful in broilers due to their short life and the lengthy withdrawal period).

  There is a need for tools to evaluate product efficacy (not just chemicals but also natural oils and plant extracts).

  It would be advantageous to have methods that can rapidly evaluate the resistance of field isolates to select the best control programmes.

  Alongside this, tools to identify subclinical coccidiosis that facilitate treatment at that time to reduce economic loss (broilers).

• Future therapy

  Fundamental research to discover biochemical pathways in Eimeria that could be targeted for drug treatment is desirable.

  Such research is long term but could, if pursued, eventually lead to better prevention and treatments for coccidiosis.

  No new drugs have been introduced for ~ 30 years but new combinations of existing drugs continue to be developed and marketed.

  GAPS :

  Need to more fully understand parasite specific biochemistry that could be exploited for novel anticoccidial drug development.

• Commercial potential for pharmaceuticals in Europe

  Despite this, anticoccidial drugs remain the main products for controlling coccidiosis.

  Developing new products will depend on national or regional policies regarding use and regulation of anticoccidial drugs. It will require continued engagement of veterinary pharma and the availability of large-scale distribution chains.

  GAPS :

  Registration of new anticoccidial feed additives is expensive and takes a long time.

  Much current research is on the evaluation of natural products (oils, plant extracts) but until recently there has been no evidence of commercial interest.

  It would be helpful to develop models on sustainability of poultry production using alternative ways of production.

• Regulatory and/or policy challenges to approval

  In Europe EFSA evaluates safety and efficacy of prophylactic anticoccidial drugs; this is very thorough and covers all aspects of use. Two major groups representing veterinarians in Europe (FVE and PSVG) agree that in-feed or in-water prophylactic use as well as for treatment is a necessary option for rearing short-lived birds such as broilers, as well as for turkeys where there is no licenced vaccine. However, retailers, acting on the concerns of consumers, may change the current landscape in terms of anticoccidial use. Campaigns around animal welfare (growth rates) as well as increased preference for products labelled as “No Antibiotics Ever” and “Raised without Antibiotics” might impact on the use of registered products. This is happening in the USA where ~ 40% of the broiler market is now using vaccination either alone or in combination with anticoccidial drugs.

• Commercial feasibility (e.g manufacturing)

  High investment costs to register or maintain registration of anticoccidials has led to the disappearance of several drugs; most importantly this has had negative impacts on turkey production.

  In some settings (eg organic) anticoccidial feed additives cannot be used, although they can be used therapeutically in the case of disease outbreaks.

  GAPS :

  Consumers and retailers, especially in the USA where these are categorised not as feed additives but as antimicrobial drugs, seem increasingly be against the use of anticoccidials. This reduces the likelihood that private companies will invest in new molecules. In the EU there is also a growing interest in No Antibiotics Ever production which could in future have an impact on the prophylactic use of the ionophores.

• New developments for diagnostic tests

• Requirements for diagnostics development

  Diagnostic tests that could be conducted directly on farms or in basic laboratories without the need for PCR equipment to distinguish species would be helpful. PCR and qPCR methods for Eimeria are hard to adopt.

  There is little information yet on the global distribution of the three more recently identified species of Eimeria that can infect chickens and no rapid tests.

  Differentiating vaccine parasites from field strains remains difficult or impossible.

  There are opportunities to be able assess the impact of subclinical disease on performance.

  GAPS :

  Impacts on bird health and growth are not solely related to the species of Eimeria that infect the intestinal tract but also to the numbers of oocysts ingested, the immune status of the bird and its general health: identification of species alone is not enough.

  Tests that could distinguish non-pathogenic carriage from subclinical coccidiosis would be very useful but difficult to develop. Would benefit from collaboration across academic/commercial sectors – to understand the impacts of subclinical disease and whether there is a need for such tests.

  Rapid molecular assays are needed for surveillance of the three newly identified species (Eimeria Iata (OTU-X), Eimeria nagambie (OTU-Y), and Eimeria zaria (OTU-Z).

  DIVA molecular assays to distinguish infections due to field or vaccinal parasites.

  Rapid tests to identify non-pathogenic carriage and subclinical coccidiosis.

• Time to develop new or improved diagnostics

  Assays for new species could be developed fast; assays for strains/DIVA or to differentiate drug resistant/susceptible, or simple carriage/subclinical disease would take much longer.

• Cost of developing new or improved diagnostics and their validation

  Hard to estimate but likely to be relatively expensive as most validation would require some return to classical methods (parasite purification/cloning by limiting dilution/assessment of clinical lesions etc). The cost of equipment needed to perform rapid throughput diagnostic tests would also be relatively high.

• Research requirements for new or improved diagnostics

  Horizontal studies are fundamental to determine intra-specific variability and association with specific phenotypes. Parasite collections of samples isolated in different countries would be of great benefit, if available to the scientific community.

  GAPS :

  A centralised repository of defined laboratory strains and field isolates that is available to the research community would be a valuable resource and should also include data on pathogenicity, elimination profiles, survivability of oocysts in litter under different conditions etc.

• Technology to determine virus freedom in animals

  Not applicable. All animals reared on the ground are at risk of infection and may become carriers.

• New developments for vaccines

• Requirements for vaccines development / main characteristics for improved vaccines

  Current live anticoccidial vaccines are intended to induce host immune responses that minimise or prevent replication by Eimeria species parasites in the event of subsequent challenge. They are effective in well controlled environments but less so in situations that do not allow re-cycling of parasites.

  Any new anticoccidial vaccine must protect against clinical coccidiosis and minimise the economic and welfare impacts of subclinical coccidiosis without compromising efficiency of production. However, it does not need to be 100% effective in reducing parasite replication.

  Optimally, new vaccines should be capable of inducing immune protection against challenge without the need for recycling of oocysts.

  Ideally vaccines would induce immune responses that cross-protect against more than one species of parasite.

  GAPS :

  Attenuated live vaccines remain unavailable for turkeys and other poultry species.

  There is a need for more vaccines that can induce robust protection faster than three weeks for use in broilers (short living) and to protect young birds in environments where there is a heavy load of contaminating parasites.

  Subunit anticoccidial vaccines are currently not commercially available for any species.

  More robust subunit or recombinant vectored vaccines are needed that are suitable for mass administration to flocks.

  Studies on adjuvants are needed to investigate how protective immune responses can be maximised, especially to potentiate cellular immunity.

  In vitro assays that are predictive of in vivo immunoprotection (correlates of protection) are highly desirable.

• Time to develop new or improved vaccines

  The robust nature of current live vaccines is reflected in the continuing entry into the market of these kinds of vaccines. Successful development requires high quality animal and laboratory facilities, a supply of SPF chickens and strict biological control to prevent cross-contamination. It may take from several months to several years to develop seed stocks depending on the number of species targeted and whether parasites are attenuated or not.

  Next generation recombinant or subunit vaccines depend on identification of effective vaccinal antigens. A small number of antigens from up to three species have been subject to testing in experimental animal systems, using various methods of delivery. The time from these early ‘proof of concept’ outcomes to a commercial vaccine is likely to be 10-15 years.

  GAPS :

  Few effective vaccinal antigens have been identified to date. The number of antigens required in an effective subunit vaccine remains unknown.

  Better vaccine delivery strategies are required, especially those based around live, replicating vaccine ‘vector’ platforms that induce a broad range of immune responses.

• Cost of developing new or improved vaccines and their validation

  Costs of developing and validating new live anticoccidial vaccine formulations is relatively high given the requirement for in vivo parasite selection, amplification and validation.

  Costs of validating recombinant or subunit anticoccidial vaccines is likely be high in the absence of in vitro assays that can accurately predict protective capacity.

  GAPS :

  Eimeria species parasites cannot efficiently complete their lifecycle in vitro. Improved protocols supporting in vitro parasite amplification would significantly reduce the cost of vaccine development and live parasite vaccine production.

  Appropriate correlates of immune protection for in vivo or in vitro vaccine development are urgently required.

• Research requirements for new or improved vaccines

  Protocols supporting the selection of precocious parasite lines as the basis of attenuated live vaccines are well established. The development of recombinant or subunit vaccines will depend on identification of immunoprotective antigens and commercial validation of one or more effective delivery strategies (e.g. DNA, protein, nanoparticles or vectored vaccine delivery). Where appropriate one or more adjuvants may be required.

  The cost of manufacturing a new or improved vaccine must be sufficiently low for it to effectively compete with anticoccidial drugs. Similarly, low-cost, low-labour route of administration will be required with scope for automation.

  GAPS :

  Attenuated lines of Eimeria species that infect the turkey and other poultry species are required for development of new live vaccines.

  Few effective vaccinal antigens have been identified to date. The ability of these antigens to induce immune protection in genetically diverse outbred hosts under field conditions is yet to be determined.

  Research into effective adjuvants is required, especially to induce cellular immune responses.

• New developments for pharmaceuticals

• Requirements for pharmaceuticals development

  For registration of anticoccidial drugs in the EU, EFSA currently evaluates the submitted dossiers. Since the beginning of 2021 they have operated a transparency policy, which is complicating applications. For synthetic products, where the potential for use and sales in Europe is limited, strategic decisions are being made by some companies to no longer invest in developing or re-registering these products (which means disappearance of products from the market).

• Time to develop new or improved pharmaceuticals

  The registration process is long and can be unpredictable.

• Cost of developing new or improved pharmaceuticals and their validation

  The investment costs for bringing new products to the market are very high. This has and will cause a decrease in available products.

• Research requirements for new or improved pharmaceuticals

  A priority is to carry out fundamental research into target discovery, using machine learning and AI approaches to interrogate the large amount of ‘omics data on coccidial parasites that has been generated in the past decade.

Disease details

• Description and characteristics

• Pathogen

  Coccidiosis is a generic term that denotes disease caused by a subclass of apicomplexan protozoan parasites commonly called Coccidia. They are classified under phylum Apicomplexa; suborder Eimeriorina; family Eimeriidae.

  Coccidiosis of chickens, turkeys, other poultry hosts, and many species of mammalian livestock is caused by different species of the genus Eimeria.

  More than a single Eimeria species may co-infect each host, and mixed infections are a common finding in field samples.

  GAPS :

  Interactions between species found in mixed infections in the chicken are not well understood.

  Little is known concerning life cycles, immunogenicity, pathogenicity, and pathological consequences of Eimeria infections in turkeys and other poultry.

  Understanding the contribution of Eimeria infection to dysbiosis is incomplete.

• Variability of the disease

  Most Eimeria species are strictly host specific with the exceptions in poultry are the turkey species Eimeria dispersa, which can also infect quail and pheasants, and Eimeria innocua and E. adenoeides which can infect grey partridge and northern bobwhite.

  The life cycles of all Eimeria species are monoxenous with no intermediate hosts.

  Each Eimeria species has a predilection for a particular region of the gut (from duodenum to colon, including the caeca) where sporozoites invade and endogenous stages replicate and differentiate within epithelial cells.

  Disease severity is variable and depends on many factors including the Eimeria species involved, virulence of the strains, infection load, breed, age and immune status of the host.

  GAPS :

  Factors involved in intestinal site specificity are poorly understood.

  Recent work has indicated that the extent and relevance of intra-specific genetic diversity is wider than previously thought.

  Three new Eimeria species have been described in chickens but their occurrence and intraspecific variation have not been studied.

• Stability of the agent/pathogen in the environment

  Oocysts shed in the faeces are relatively stable in the environment and can maintain viability from weeks to months; life expectancy is much shorter in commercial poultry houses. High humidity and mild temperatures favour viability, whereas high temperatures, low moisture and high concentrations of ammonia, as found in reusable litter, can rapidly kill the parasites.

  The oocyst wall is composed of multiple layers with distinct chemical compositions, and constitutes an efficient barrier to chemical agents, including many commonly used disinfectants. Several disinfectants claim to decrease Eimeria oocyst load in the environment. Physical agents such as heat and abrasion can rapidly kill oocysts.

  GAPS :

  Development of disinfectant oocysticides that can be used in the presence of animals would be beneficial.

• Species involved

• Animal infected/carrier/disease

  Seven well recognised species of Eimeria have long been known to infect chickens: E. tenella, E. acervulina, E. maxima, E. necatrix, E. brunetti, E. praecox and E. mitis.

  Recently, the isolation and detailed molecular and biological characterisation of parasites representing three cryptic genotypes (previously termed operational taxonomic units, OTU) revealed these to be independent species that infect chickens: E. Iata (OTU-X), E. nagambie (OTU-Y) and E. zaria (OTU-Z). All three of these are common across the southern hemisphere and have also been detected in North America and southern Europe.

  Two further potential species have been reported but their status remains doubtful as isolates are not available for robust characterisation. These are E. mivati (from 18S RNA data this is likely synonymous with E. mitis although it is mentioned as an included species in two vaccines) and E. hagani (although previously part of one vaccine, this parasite was described just once in 1938).

  In turkeys seven species have been described: E. adenoeides, E. gallopavonis E. meleagrimitis, E. dispersa, E. innocua, E. meleagridis and E. subrotunda. Recent studies do not report E. subrotunda.

  In other poultry and mammals there is a myriad of species described. In guinea fowl and red partridge, some species of ancient description have been validated in the last 20 years.

  GAPS :

  Few details are known of the life-cycles and phenotypes of four species of turkey coccidian.

  Knowledge of coccidian species in other hosts is limited.

  There is high genetic diversity between strains/isolates of species. This is not fully defined for all species, but it does pose potential risk that vaccination may select for resistant populations. For example, within E. maxima strain diversity is known to cause antigenic divergence which can lead to lack of immune cross-protection between strains. For this reason, some anticoccidial vaccines contain more than one strain of the same species.

  There has been uncertainty about turkey species taxonomy and identification, but recent research has clarified this and provided new tools for species identification.

• Human infected/disease

  Poultry coccidia are host-specific and do not normally infect additional hosts, including humans.

  GAPS :

  Host specificity and evolutionary origins of Eimeria are still open questions. There are speculations that some Eimeria species seen in chickens and turkeys originally came from foreign hosts.

• Vector cyclical/non-cyclical

  Parasites have a monoxenous life cycle with no cyclical vectors.

  Arthropods, wild birds, rodents, humans and any other animals with access to poultry faeces may act as mechanical vectors of infective oocysts.

  GAPS :

  Global spread of Eimeria species, especially the newly discovered species (E. lata, E. nagambie and E. zaria) is not well understood, for example, is there a role for global trade?

• Reservoir (animal, environment)

  Wild jungle fowl are known to host Eimeria that appear to be the same species as those described in the domestic chicken.

  In the EU free-range populations of Gallus gallus may transmit infections, but as they suffer the disease and are the same host species, this is not defined as a reservoir.

  Oocysts can survive in the environment for long periods especially when contained within poultry faeces/manure.

  GAPS :

  There has been little research on how Eimeria oocysts transmit between premises.

• Description of infection & disease in natural hosts

• Transmissibility

  Transmissibility via the faecal-oral route is high; it is very rare to find commercial flocks not infected with coccidia. Contact of hosts with faeces containing sporulated oocysts is the most frequent source of infection. All poultry are at risk and it is impossible to prevent susceptible birds getting infected.

  Eimeria parasites have been known to appear in new poultry houses immediately after build; it is suspected that these are introduced through contaminated water.

  Other infection sources may include contaminated feed, old litter, poultry-rearing equipment, human agency (dirty hands or boots), and non-host animals transporting live oocysts (birds, rodent vermin, flying insects, other invertebrate pests).

  GAPS :

  Few safe, effective oocysticides exist and more effective, safer administration methods need to be developed particularly to overcome interference by organic matter in litter and to improve penetration of irregular surfaces.

  Cleaning with effective disinfectants or detergents should be improved to mechanically reduce parasite loads on the ground and on equipment (feeders and drinkers).

• Pathogenic life cycle stages

  All poultry coccidia are monoxenous and most are host-specific. The life cycle has two phases: endogenous (in the host’s gut) and exogenous (outside the host). Each species invades and develops within specific parts of the intestinal tract.

  Parasites are haploid through the life cycle, except for zygotes and unsporulated oocysts, which are diploid. The endogenous phase begins with ingestion of live, sporulated oocysts. These are mechanically disrupted in the gizzard, releasing sporocysts, from which sporozoites excyst under the action of trypsin and bile salts in the duodenum. Freed sporozoites migrate through the intestine to their preferred site where they actively invade enterocytes. Each intracellular sporozoite transforms into a trophozoite which undergoes several rounds of mitosis and enlargement to produce a multinucleate schizont which divides by schizogony to produce large numbers of daughter merozoites. Released merozoites invade fresh epithelial cells, initiating the next phase of asexual schizogony and so on. Each species completes a specific number of rounds of schizogony (usually up to four) before gametogony, whereby the invading merozoites give rise to either a female macrogametocyte, which develops into a single macrogamete (ovum), or a male microgametocyte, which divides to produce many microgametes (spermatozoa). Microgametes fertilize the macrogametes, and the resulting diploid zygotes develop into immature unicellular oocysts (at this point non-infective), which are expelled from the host in its faeces.

  The exogenous phase involves sporulation (internal division) of expelled non-infective oocysts to produce sporulated oocysts, each containing four sporocysts that in turn contain two sporozoites. The first division is meiotic, subsequent divisions are mitotic. Sporulation requires adequate levels of environmental warmth, moisture and oxygen. Oocysts are not infective unless sporulated.

  GAPS :

  Little is known about control of parasite replication, gene expression and stage differentiation which strongly influence virulence, pathogenicity and attenuation.

  Examination of field populations indicates high levels of polymorphism and mixed infections however nothing is known about the impact of this on the selection of novel strains with changed virulence or pathogenicity, or on gamete ratios during the sexual phase of the life cycle.

  The frequency of cross-fertilisation between genetically distinct strains of an Eimeria species is largely unknown.

• Signs/Morbidity

  Naïve birds of any age are susceptible to infection. Three levels of disease severity are recognized: 1) coccidiasis, a mild infection causing no adverse effects, 2) subclinical coccidiosis, causing slight but economically important reductions of host growth and feed utilization, 3) clinical coccidiosis, a serious (perhaps fatal) disease.

  Disease severity is affected by infection load and immune status of the host. The least virulent coccidian species cause diarrhoea, poor skin pigmentation, morbidity, reduction of weight gain and poor feed conversion. The more virulent coccidian species can produce similar signs and, with heavier infections, various degrees of gut haemorrhage and perhaps death.

  Birds can become infected without any disease (coccidiasis) or without diagnosis and the parasites appear rapidly after clean-outs of litter; newly hatched chicks placed into houses can become exposed to parasites very rapidly either from the litter or from parasites being excreted by infected litter mates.

  GAPS :

  Little is known of specific virulence factors within and between parasite species.

  Virulence differences may be important between isolates of the same species.

• Incubation period

  Sporulation of oocysts typically can take as little as <24 hours for some species in ideal conditions but this extends to perhaps up to 3 days under field conditions. The endogenous phase in birds is short, about 4.5-6 days, and is known as the prepatent period, i.e. the time that elapses between ingestion of sporulated oocysts and earliest excretion of progeny unsporulated oocysts.

• Mortality

  Some species (Eimeria tenella, E. necatrix, E. brunetti and E. maxima in chickens; E. adenoeides, E. gallopavonis, E. meleagrimitis and E. meleagridis in turkeys) can cause moderate to massive mortality in naïve birds. During disease outbreaks, birds that lack acquired immunity are highly susceptible which can result in high mortality. Deaths in commercial flocks are usually mitigated and reduced by birds having acquired immunity either through vaccination, or due to early life exposure to low numbers of parasites.

  Other species never (or very rarely) cause deaths (E. acervulina, E. mitis and E. praecox in chickens; E. dispersa, E. innocua, and E. subrotunda in turkeys).

  GAPS :

  Few details are available for the three newly identified species that infect chickens (E. lata, E. nagambie and E. zaria).

• Shedding kinetic patterns

  Once a flock is infected, accumulation of oocysts in the litter proceeds near continuously as parasites are ingested and shed asynchronously. In generally, litter oocyst counts peak at ~ 3-5 weeks in chickens and ~4-6 weeks in turkeys but there is no hard-and-fast rule. Several peaks may be observed, caused by co-infection with multiple species. Eventually litter oocyst counts decline, due to acquired host immunity which limits shedding and adverse conditions for oocyst survival in litter.

  In breeding and egg-production flocks there is long-term reciprocity between oocyst production and immunity. Once protective immunity is acquired it is not lost, but complete cessation of oocyst production almost never occurs resulting in enzootic stability.

  GAPS :

  Epizootiology research is greatly hampered by an inability to distinguish strains within species.

  Modern broiler hybrids appear to be more ‘resilient’ to coccidiosis than older breeds and can thrive despite high levels of parasite infection and replication. This may be linked also to improved feed quality and environmental conditions; whatever the reason, litter oocysts in modern broiler flocks can reach high numbers very rapidly.

• Mechanism of pathogenicity

  The main pathology is due to destruction of gut epithelia, villous atrophy and for some species, disruption of sub-epithelial tissues.

  Epithelial destruction and villous atrophy by those species that infect the small intestine cause malabsorption of essential nutrients, reduced activity of digestive enzymes and leakage of plasma proteins. Infection also induces increased mucous production, which further compromises nutrient absorption. Other poorly understood mechanisms cause reduced feed and water intake; reduced weight gain and feed conversion; increase of gut acidity leading to longer gut passage time and poor uptake of vitamin A and xanthophylls; reduction of digesta viscosity. These traits are all associated with poor welfare and performance.

  Sub-epithelial disruption by E. tenella and E. necatrix in the caecum, and occasionally by E. brunetti in the terminal ileum, caecum or rectum can result in severe haemorrhage in chickens causing pain, prostration and death.

  Gut epithelial damage by coccidia and the resulting protein leakage also renders hosts more susceptible to infection by the gram-positive bacterium Clostridium perfringens, which causes necrotic enteritis.

  GAPS :

  To control intercurrent coccidiosis and necrotic enteritis, there is considerable potential for the discovery of feed additives to improve gut integrity.

  Little is known of specific virulence factors.

  The role of probiotics in balancing the intestinal flora and having a beneficial impact is poorly understood.

  Little is known of the interactions of Eimeria and Eimeria-induced host responses with other co-infecting enteric pathogens including Salmonella enterica, Campylobacter jejuni, Clostridium perfringens, Escherichia. coli and enteric viruses.

• Zoonotic potential

• Reported incidence in humans

  There are no reliable reports of infection in humans by poultry coccidia.

  Indirect zoonotic potential includes the known association of E. tenella infection with increased caecal colonisation and shedding of Campylobacter jejuni, a leading cause of foodborne bacterial zoonoses.

  GAPS :

  Controlling coccidiosis may have a positive effect on controlling spill over infections from E. coli, non-typhoidal serovars of Salmonella enterica, and species of Campylobacter.

• Risk of occurence in humans, populations at risk, specific risk factors

  No risk since poultry coccidia are extremely host specific.

• Symptoms described in humans

  Not applicable.

• Likelihood of spread in humans

  Not applicable.

• Impact on animal welfare and biodiversity

• Both disease and prevention/control measures related

  Poultry coccidiosis is exacerbated in intensively reared domesticated breeds of jungle fowl (Gallus gallus) and turkeys (Meleagris gallopavo). Welfare is not normally compromised provided that chemoprophylactic and/or vaccinal control remains effective.

  Details on current vaccines and anticoccidial drugs are given in section 2: Vaccines availability and section 3. Pharmaceutical availability.

  In brief, vaccine use is widespread in breeding and layer flocks and enhances welfare, as would much less intensive rearing if introduced in the future. Live non-attenuated vaccines may occasionally cause clinical disease. Live vaccines induce species-specific immunity so can be targeted to problem species without reducing parasite diversity.

  Chemoprophylaxis is widely used; for broilers it protects them from clinical disease but may be compromised by drug resistance. There are no known welfare implications and unlike vaccines, anticoccidial drugs are active against all species of coccidia.

  The possibility of banning anticoccidial drugs is much discussed in Europe and could adversely affect welfare unless vaccines became more widely used for broilers.

  Banning ionophores for broilers could also compromise control of necrotic enteritis for two reasons; first ionophores have some (variable) activity against Clostridium perfringens and second coccidial infections predispose birds to clostridial attack so failure to control coccidia will exacerbate necrotic enteritis.

  Wet litter resulting from dysbiosis caused by some species of Eimeria poses a significant risk for pododermatitis, hock and breast burn, all conditions with serious welfare consequences.

  GAPS :

  Although pharmaceutical companies are discouraged from seeking new anticoccidial drugs, if drug screening were to continue a reliable in vitro testing system would be required.

• Endangered wild species affected or not (estimation for Europe / worldwide)

  Wild jungle fowl in Asia and turkeys in North America are the natural hosts of the same coccidia that cause commercially important diseases. They remain susceptible to these species and little is known of the epidemiology of current wild bird infections, particularly whether domesticated birds are a source of infection for wild birds, or vice versa. As wild bird densities are much lower than on farms, the latter risk is low.

  GAPS :

  There is a general lack of knowledge of parasite intraspecific variation, population structure and epidemiology.

  Recent work indicates that genetic variation is greater than previously recognised and that parasite population structure and epidemiology differs according to geographic location and host density. The impact of this on sustainability of control is not known.

• Slaughter necessity according to EU rules or other regions

  No.

• Geographical distribution and spread

• Current occurence/distribution

  Distribution of the well-known species is worldwide.

  Newly described species of chickens (E. lata, E. nagambie and E. zaria) have mainly been described in the southern hemisphere but also reported in North America and southern Europe.

• Epizootic/endemic- if epidemic frequency of outbreaks

  In intensively reared poultry and those reared with access to the external environment, coccidiosis can occur at any time of year.

• Speed of spatial spread during an outbreak

  In poultry reared on litter infections, if not controlled, can spread rapidly within a flock especially if there are high stocking densities. Environmental conditions, such as high moisture levels in the litter, can exacerbate coccidiosis.

  Because parasites are highly infectious and grow fast, outbreaks generally do not last for long periods; oocysts shed by the first infected birds become infectious 1-3 days later, by which point clinical signs from these first birds are resolving and immunity is developing. More birds will be infected in the second round, the absolute numbers depending on the initial burden of oocysts in the henhouse and the number of birds ingesting them. Most outbreaks of clinical disease happen during the third or fourth week of life, suggesting that a critical threshold is reached at that time; thereafter there are few naïve hosts available.

• Transboundary potential of the disease

  Oocysts from infected birds can remain in buildings and be carried by mechanical means (equipment, clothing, insects, other animals and wild birds) so there is transboundary potential, although parasites are already ubiquitous. Poultry houses acquire parasites rapidly and preventing infection is difficult wherever poultry are raised even in standard contained laboratory facilities. Spread of new Eimeria species throughout the southern hemisphere in the past decade indicates huge potential for transboundary spread.

  GAPS :

  There is a general lack of knowledge of parasite intraspecific variation, population structure and epidemiology.

• Route of Transmission

• Usual mode of transmission (introduction, means of spread)

  Eimeria parasites that cause coccidiosis in poultry have an oral-faecal life cycle. The most common transmission is through birds ingesting oocysts from contaminated litter or any other surface contaminated with infected faeces such as feeders and drinkers.

  GAPS :

  Modern broiler genotypes appear to support high levels of parasite replication so there is potential for much higher levels of contaminated litter/ground. This could be attributed potentially to more subclinical infections (that are not detected) or be linked to increases in drug resistance.

• Occasional mode of transmission

  Experimentally infections can be initiated by intravenous and cloacal infection of chickens with asexual (sporozoites/merozoites) stages but these are not known to occur naturally.

• Conditions that favour spread

  High stocking density in warm, wet conditions favour the sporulation of oocysts and facilitate higher levels of infection. However, dry litter does not reduce risk to zero; for example, guinea fowl are raised in dry and dusty conditions but suffer significant coccidiosis.

  GAPS :

  Common in developing countries where there are high risks of ‘new’ genotypes emerging.

• Detection and Immune response to infection

• Mechanism of host response

  In general, immune responses to Eimeria infection include cellular and humoral components, and lead to protection against re-infection in a species or strain-specific manner. Immunity to one Eimeria species does not confer immunity to other species of Eimeria, nor are different strains of the same species equally protective against that species.

  In chickens, cell-mediated immunity plays a major immunoprotective role during primary infection and re-infection. Whilst CD4+ αβ T cells are essential for immunoprotection in primary infections with at least E. maxima and E. tenella, the requirement for specific cell subsets and effector mechanisms is less stringent for immunoprotection against re-infection.

  Chickens infected with Eimeria parasites produce both local and systemic antibodies, which can play a role in protection provided they are present at high levels.

  In turkeys, primary infection of poults with Eimeria adenoeides induces upregulation of CD4+ and CD8+ T cells as well as an elevated ratio of CD4+ to CD8+ T cells, leading to the development of protective immunity.

  GAPS :

  Many facets of innate and adaptive immunity to coccidiosis are not well defined including the role of molecular pattern recognition; the cells, molecules and pathways involved in early stages of the protective immune response; essential components of immunoprotection; and the influence of host genotype, infection history and parasite dose.

  Large numbers of parasite molecules are immunogenic but not immunoprotective.

  There is a need to understand immunoprotective responses that operate after both primary and second infection and to differentiate immunoprotective redundancy from the cumulative effects of responding to multiple immunoprotective components.

• Immunological basis of diagnosis

  There are no serological assays in routine use for detection of Eimeria infection in chickens and turkeys or for differential diagnoses between infections caused by different strains or species of parasites.

  Convalescent animals produce antibodies and T-cells that react with many parasite-derived epitopes, including some that cross- react between strains and species of Eimeria. However, the overall magnitude and breadth of the immune response does not correlate with protective immunity and there are no in vitro assays that can be used as a correlate for successful vaccination.

  GAPS :

  Identification of in vitro correlates of protection for new vaccine development.

  Research into cellular immunity of chickens, particularly knowledge on memory cells would be important when considering protection of long-lived birds.

  As infection is acute and established after first contact with the parasite, most specific immunological markers are detected too late.

• Main means of prevention, detection and control

• Sanitary measures

  The aim is to minimise background levels of oocysts so they do not overwhelm vaccination or chemoprophylaxis of subsequent flocks. Complete elimination is not possible and low levels of oocysts are generally accepted as being helpful to build flock immunity. It is crucial to keep moisture levels as low as possible; as oocysts are shed in droppings, which are moist, even dry litter does not prevent risk.

  Biosecurity measures include changing footwear and cleaning equipment between animal facilities, arthropod and rodent control and general hygiene. Because oocysts are resistant to most disinfectants, footbaths are generally not effective.

  Litter change between flocks is practised in EU and decreases the starting/background oocyst levels but conversely fresh litter provides excellent conditions for sporulation and oocyst survival.

  In the US litter is reused for successive broiler flocks and may become composted which enhances oocyst degradation and lowers background infection levels. When built-up litter is cleared (usually annually) washing and disinfection must be assiduous to prevent carry-over of oocysts that new litter conditions will not suppress.

  Rearing birds on wire mesh, which is less prevalent in Europe, means that droppings fall through wire, reducing recycling of infection.

  GAPS :

  There is a need for improved disinfectants (see also Section “Description and characteristics – Stability of the agent in the environment” and “Description of infection and disease in natural hosts – Transmissibility”).

  Rigorous methods for cleaning with disinfectants and detergents must be improved to mechanically remove oocysts from buildings; this includes cleaning of equipment. Epidemiological surveys often indicate that this is not done thoroughly.

  Higher levels of oocysts in the environment could be due to more subclinical infections or increasing levels of drug resistance.

• Mechanical and biological control

  Mechanical control (changing litter and washing buildings and equipment) as described in Section “Sanitary measures”.

  Biological control includes vaccination as detailed in Section “Vaccines availability” and Section “New developments for vaccines”.

• Prevention through breeding

  There is some evidence that modern hybrid birds (broilers and layers) are more resistant to clinical disease than older breeds.

• Diagnostic tools

  Most cases of coccidiosis are subclinical; clinical coccidiosis may be signalled by observing behaviours (prostration, ruffled feathers) or seeing diarrhoea or bloody faeces.

  In the field the presence of Eimeria acervulina, E. necatrix and E. tenella can be determined by observing intestinal lesions, which can be corroborated by microscopic examination of intestinal smears (although some lesions remain after oocysts have gone). Other species may be diagnosed by combinations of abnormal intestinal contents, petechiae, general congestion and smears.

  Species tropisms are as follows:

  Duodenum - E. acervulina, E. praecox, E. maximaJejunum /ileum - E. maxima, E. mitis, E. necatrix (asexual stages)Caeca - E. tenella, E. necatrix (oocysts)Ileum/colon/rectum/base of caeca – E. brunetti.

  The most easily observed and most damaging stages are the schizonts of E. tenella and E. necatrix; and the gametocytes and oocysts of the remaining five species.

  As described in Section “Diagnostics availability”, rapid tests are developed that can discriminate all seven well known species that infect chickens, but tests are not commercially available.

  GAPS :

  Need for rapid diagnostic tests for strain differentiation, including to distinguish drug resistance/sensitivity and vaccine strains from field strains.

  Tests to diagnose new species E. lata, E. nagambie and E. zaria (formerly OTU genotypes) are required.

• Vaccines

  See Section “Vaccines availability” and “New developments for vaccines”.

• Therapeutics

  See Section “Pharmaceutical availability” and “New developments for pharmaceuticals”.

• Biosecurity measures effective as a preventive measure

  See Section “Sanitary measures”.

• Border/trade/movement control sufficient for control

  Not considered appropriate since the disease is endemic worldwide.

• Prevention tools

  Prevention of coccidiosis combines good biosecurity and sanitary practices (including disinfectants to lower environmental load) with vaccines and/or prophylactic use of anticoccidials.

• Surveillance

  Coccidiosis is not a reportable disease, but surveys of geographic presence of species have been conducted by quantitative PCR and conventional parasitological methods.

  Utility of such surveys can aid in increasing confidence of using appropriate vaccines. Extensive surveys have been conducted around the world to determine the drug sensitivity of field isolates of Eimeria. These surveys have often been undertaken to aid poultry producers in the selection of appropriate drugs for inclusion in poultry feeds.

  GAPS :

  The genetic basis of resistance to anticoccidial drugs is unclear, thus molecular surveillance is currently not possible. Instead this requires relatively expensive low-throughput in vivo screening.

• Past experiences on success (and failures) of prevention, control, eradication in regions outside Europe

  Coccidiosis is endemic and not appropriate for eradication. Most countries have control measures in place by means of the use of anticoccidial drugs, vaccination or both.

  GAPS :

  Failures can occur due to drug resistance. Some anticoccidial feed additives have been withdrawn due to very rapid resistance in field conditions.

• Costs of above measures

  The greatest costs associated with control are due to prophylactic and therapeutic control of broiler coccidiosis. See section “Direct impact on (a) production” and “Direct impact on (b) cost of private and public control measures”.

• Disease information from the WOAH

• Disease notifiable to the WOAH

  No.

• WOAH disease card available

  No.

• WOAH Terrestrial Animal Health Code

  Not applicable.

• WOAH Terrestrial Manual

  Not applicable.

• Socio-economic impact

• Zoonosis: impact on affected individuals and/or aggregated DALY figures

  Humans are not susceptible to poultry coccidia.

  There is some evidence that coccidiosis may indirectly affect food safety due to high burdens of parasites than can compromise the integrity of the chicken gut, enhancing colonization by bacteria including zoonotic agents. Gut material compromised by coccidiosis may be more susceptible to damage at the processing plant thereby increasing contamination with intestinal contents including zoonotic agents.

  In addition, human livelihoods are affected by poultry coccidiosis especially in low-and-middle income countries where people are dependent on “back-yard” poultry for meat and eggs and for generating income.

  GAPS :

  There are no good recent models of the economic costs of coccidiosis across different parts of the world.

  There is a need to understand the effect of coccidiosis on the spread and behaviour of other avian gut pathogens, including zoonotic agents.

• Zoonosis: cost of treatment and control of the disease in humans

  Not applicable.

• Direct impact (a) on production

  Coccidia are ubiquitous and occur in wild bird hosts from which domesticated poultry were derived, and everywhere that their descendants are reared. The threat of disease increases with greater intensiveness of rearing in countries with high poultry commercialization. There are also risks in low-and-middle income countries where poultry rearing is intensifying rapidly with variable access to sophisticated husbandry and control methods.

  Accurate estimates of the cost of coccidial diseases worldwide are difficult to obtain.

  Recent estimates indicate that the UK poultry industry incurred additional costs of GBP99.2 million in 2016 due to the consequences of Eimeria infection and costs of control. Of this, 83.1% was attributed to the economic impacts of disease morbidity. Extrapolation to the European poultry industry suggests costs of GBP1.02 billion in 2016. Costs calculated in Brazil, Egypt, Guatemala, India, New Zealand, Nigeria and the USA were used along with those from UK and Europe to estimate a global cost of ~ £10.4 billion at 2016 prices (£7.7–£13.0 billion), equivalent to £0.16/chicken produced.

  In the total absence of anticoccidial control the median impact of infection was found to peak at between EUR2.55 and EUR2.97 in lost production per meter squared of broiler house over a 33-day growing period.

  Poultry producers rate coccidiosis as the number 1 disease that affects production.

  GAPS :

  Cost models need to be regularly updated and expanded to other parts of the world with rapidly expanding poultry production and/or dependency on backyard flocks.

  Costs of subclinical coccidiosis may be under-evaluated because it is not diagnosed as a unique cause of losses as many other pathogens can impact on profitability.

  Indirect costs of Eimeria infection are unclear, including necrotic enteritis, enteric dysbiosis and the consequences of poor litter quality caused by coccidiosis.

• Direct impact (b) cost of private and public control measures

  Based on the same data as in Section 18.4 (a) “Direct impact on production”, the costs of control measures through drug prophylaxis and vaccination, and chemotherapeutic treatments amounted to 16% (GBP15.9 million) of total costs of disease and its control in the UK in 2016. Equivalent data for other parts of the world are potentially very variable, depending on local conditions with examples including GBP158.9 million in the USA and GBP125.1 million in Brazil.

  Given that coccidiosis is the major condition that affects productivity of poultry, it is likely to have a much higher direct impact that currently recognised.

• Indirect impact

  The worldwide poultry market is extremely adaptable, and local problems with coccidiosis are unlikely to have major widespread indirect effects. However, in recent decades, poultry meat has become the principal source of animal protein both in high income and low-and-middle income countries and has a crucial role in sustaining human health. This has been achieved by intensifying poultry production, which would be compromised if it were not possible to control coccidiosis. The likely consequence of such failure would be a decline in poultry production and increased prices of meat and eggs.

• Trade implications

• Impact on international trade/exports from the EU

  Live day-old chicks are traded worldwide, but since they hatch coccidia-free and do not contact infected birds before arrival on-farm, they do not transmit disease during transport. Likewise, transport of chicken meat carries little risk of disease dissemination because carcases are eviscerated and frozen. A greater risk, although still low, is that of exported meat or eggs being contaminated with anticoccidial drug residues above the legal maximum limit.

  GAPS :

  Rapid methods for detecting anticoccidial residues in meat would aid surveillance programmes.

• Impact on EU intra-community trade

  Any ban by the EU on the use of drugs in the feed for the control of coccidiosis has the potential for causing trade disputes with those countries that use such agents and export poultry meat to the EU.

• Impact on national trade

  See Section above “Impact on international trade from the EU and “Impact on EU intra-community trade”.

• Main perceived obstacles for effective prevention and control

  The primary means of controlling avian coccidiosis are medication of the feed with anticoccidial ionophores or synthetic drugs or vaccination with live Eimeria oocysts.

  Depending on a number of factors, oocysts can remain infectious for weeks after being excreted by infected chickens, and thus serve as a source of infection to non-immune birds in the house.

  Preventing or controlling outbreaks of coccidiosis requires information on the drug sensitivity of Eimeria strains and the Eimeria species composition at the very least on individual farms. The main obstacle in the way of effective prevention is the expense and time involved in gathering data on drug sensitivity and species composition. Without this information, producers must alter treatment strategies after outbreaks occur, rather than anticipating when problems might arise.

  Another perceived obstacle is that vaccination methods may be neither efficient nor uniform in establishing high levels of immunity within 1-2 weeks of age. Attaining high levels of protection in a flock requires “cycling” of oocysts (ingestion and completion of life cycle) early during grow-out, which in turn depends upon environmental conditions that influence the rate of oocyst sporulation.

  GAPS:

  Rapid methods to identify drug sensitivity profile of individual species of Eimeria oocysts in a litter sample.

  Rapid methods to determine the immunological cross-reactivity of Eimeria strains, especially recently identified genetic variants and potentially new cryptic species.

  Development of novel methods of vaccine administration that ensure uniform exposure to vaccinal oocysts.

  Development of vaccines that do not require cycling via oocysts in the litter to achieve solid protective immunity.

  Development of attenuated vaccines for use in turkeys.

  Development of oral vaccines that do not require a cold chain.

• Main perceived facilitators for effective prevention and control

  A number of different classes of anticoccidials drugs are available, and producers are quite willing to switch from one to the other during a single growout, or between growouts.

  Producers are increasingly willing to utilize live Eimeria oocysts vaccines as a temporary or even permanent alternative to anticoccidial drugs.

  GAP: There is a need for novel anticoccidial drugs with modes of action that differ from those of existing compounds.

• Links to climate

  Seasonal cycle linked to climate

  There is some indication that coccidiosis is more frequent during wetter conditions such as during spring and autumn in the northern hemisphere. However, outbreaks of coccidiosis are reported all year round.

  GAPS :

  There is preliminary evidence that climate may impact on epidemiology of coccidiosis and influence rates of co-infection and genetic recombination of strains.

  There is a need to understand the likely future impact of climate change on pathogenicity/drug resistance/vaccine efficacy, especially in developing countries where the poultry industry is rapidly expanding.

• Distribution of disease or vector linked to climate

  The parasites occur worldwide wherever poultry are reared under intensive or semi-intensive conditions, irrespective of climate.

• Outbreaks linked to extreme weather

  There is no reason for extreme weather to have a dramatic effect, although oocysts are sensitive to below freezing temperatures over a few days and it is known that viability decreases at temperatures above 60^oC.

• Sensitivity of disease or vectors to the effects of global climate change (climate/environment/land use)

  Not known.

• Main perceived obstacles for effective prevention and control

  The primary means of controlling avian coccidiosis are medication of the feed with anticoccidial ionophores and/or synthetic drugs or vaccination with live Eimeria oocysts.

  Depending on several factors, oocysts can remain infectious for weeks after being excreted by infected chickens, and thus serve as a source of infection to non-immune birds in the house.

  Resistance to currently used in-feed anticoccidial drugs is commonly detected in field isolates. This is an obstacle to sustainability yet ionophores and combinations of ionophores and coccidiostats continue to provide a good degree of disease control. Drugs are continuously administered (usually in feed) and control programmes rely on careful monitoring and switching/shuttling of drugs to maximise performance. It is costly and time consuming to gather data on drug sensitivity and species composition on individual premises but without this information producers often end up changing treatments after disease outbreaks, rather than being able to anticipate problems before they arise.

  It is known that subclinical coccidiosis has impacts on bird welfare and efficiency of performance, but there are no methods for diagnosing subclinical disease (in live birds) and these impacts are infrequently quantified which presents an obstacle to optimising health and performance.

  If/when clinical coccidiosis emerges, there are drugs (eg amprolium, toltrazuril) that can be used in mitigation, but these have variable results and losses can still be significant, which causes high economic impact especially if outbreaks are late in the cycle. Moreover, toltrazuril cannot be used when birds are close to slaughter age because of a long withdrawal period.

  In much of the world including Europe, vaccines are rarely used in commercial broilers because of their short lifespan and potential impacts of live vaccines on performance. Here, prophylactic in feed anticoccidial drugs remain the primary method of control. Conversely, where there is pressure to produce chickens without prophylactic drugs (currently in the USA), there is reliance on vaccines, mainly non-attenuated vaccines. These kinds of vaccines are not licenced in Europe because of concerns linked to their safety. Lack of development of vaccines suitable for deployment into the mass commercial broiler market remains a key obstacle.

  In laying and breeding flocks, live vaccines (non-attenuated or attenuated) are deployed in most high-income countries and in some lower income countries. Current live vaccines induce species-specific protection and the acquisition of robust immunity even takes around 3 weeks to allow for re-cycling and re-infection with vaccine parasites which provides a boost. Thus, young birds remain susceptible to clinical or subclinical coccidiosis if they ingest high levels of sporulated oocysts, which may derive from previous flocks, or from the vaccines themselves.

  The identification of three new species of Eimeria that infect the chicken, as well as demonstration that field isolates of well-studies species exhibit high levels of genetic polymorphism and the global phenomenon of drug resistance all indicate the potential of these parasites to evolve and emerge in response to selective pressures.

  The lack of affordable diagnostics for mass use, as well as reliable modelling of the impact of poor coccidiosis control are both obstacles to accurately estimating the impact of coccidiosis.

  GAPS :

  Mechanisms of drug resistance are not known in any detail; if these were elucidated, rapid tests for resistance would be useful for surveillance/screening of isolates.

  Despite a massive increase in good quality sequence data, there is little investment in identifying and characterising potential novel drug targets for both prophylaxis and for treatment of outbreaks.

  We need diagnostics tests or biomarkers that can distinguish between disease-free carrier status and subclinical coccidiosis.

  There are no general guidelines or tools for diagnosis of the newly identified species E. lata, E. nagambie and E. zaria (formerly OTUs).

  Methods to increase production of current vaccines, for example, in vitro culture methods for species of Eimeria, would potentiate greater dissemination and use of vaccines.

  Improved understanding of the mechanisms of protective immunity would support further development of vaccines.

  Better understanding of the basis of species-specificity and lack of cross-immunity between species of Eimeria could support generation of vaccines that protect against multiple species of parasite.

  Vaccines that can be used effectively in broilers and which can compete on price with drugs would support reduction in subclinical coccidiosis.

  Identification of additional vaccine antigens from all important species, and improvements in delivery methods and adjuvants are needed.

  Collaborations between academic researchers, poultry producers and veterinary pharma are needed both to support specific projects and to share knowledge/evidence on the effectiveness of interventions and the impact of coccidiosis.

• Main perceived facilitators for effective prevention and control

  There is now a large body of molecular data defining the genotypes, transcriptomes and proteomes of important parasite species; these datasets provide excellent starting material for new approaches to diagnostics, drug discovery and antigen identification.

  There is a renewed interest from the commercial sectors (poultry industries and pharma) to work with academic researchers on projects that seek to improve background knowledge and develop new diagnostics and tools for control.

  There is increased recognition that subclinical coccidiosis has an impact on bird welfare and performance and that it can contributes to dysbiosis and necrotic enteritis as an important co-factor that potentiates invasion of the chicken gut with bacterial pathogens.

Global challenges

• Antimicrobial resistance (AMR)

• Mechanism of action

  Whilst there is knowledge of general mechanisms (eg ionophores disrupt ion balance across membranes; decoquinate inhibits mitochondrial transport) the precise basis of target specificity and selectivity of currently used anticoccidial drugs is not fully understood.

  GAPS :

  Lack of knowledge on precise molecular basis of drugs selectivity.

• Conditions that reduce need for antimicrobials

  Application of current live vaccines at day of hatch and good farm management practices to keep parasite loads low during the first three weeks of life (when immunity is not fully developed) can provide protection against clinical coccidiosis in the absence of in-feed prophylactic anticoccidials.

  GAPS :

  Lack of understanding of mechanisms of immunity, both innate and acquired.

• Alternatives to antimicrobials

  Use of live vaccines is increasing globally, mainly focused on breeding flocks and in some parts of the world layers. There are some chemical treatments that are highly specific for coccidia (eg amprolium, toltrazuril) that can be used for therapy. Increasing searches for ‘natural’ anticoccidial treatments such as essential oils and plant extracts are showing some efficacy but much lower than traditional anticoccidials.

  GAPS :

  Need to better standards for the evaluation of new kinds of prophylaxis including plant extracts, natural oils, probiotics etc.

• Impact of AMR on disease control

  Field populations of Eimeria from around the world show resistance to ionophores and to many chemicals; however, in most circumstances these drugs still have some effect at reducing the impact of disease pathology. Producers are becoming more adept at using shuttle programmes of drugs or using combination drug treatments. In high income countries vaccines may be used for several grow outs to control coccidiosis on farms with history of clinical outbreaks; this has the added benefit of introducing drug-sensitive genotypes which in turn contribute to restoration of drug efficacy.

• Established links with AMR in humans

  No clear demonstration, however resistance to ionophores could potentially co-select resistance to some antibiotics used in humans (plasmid-encoded MDR).

  GAPS :

  Need to understand mechanisms of resistance and whether this is could be co-selected with resistance to other antibiotics.

• Digital health

• Precision technologies available/needed

  GAPS :

  No digital tools are available to assist with diagnostics or control of coccidiosis.

• Data requirements

  GAPS :

  Data on geographical prevalence, species identification, drug resistance, antigenic variation and virulence of strains would be useful to target and guide control programmes.

• Data availability

  Sequencing and proteome data generated in the public and academic sectors are available in open access databases. There is some specialised curation and integration of genome, transcriptome and proteome data within ToxoDB, the Toxoplasma informatics resource that is part of the VEuPathDB project.

  GAPS :

  There is no centralised repository for non-sequence data; most ‘big data’ on pathology, epidemiology/prevalence, drug resistance profiles etc are held by big producers and a small number of companies who are developing vaccines.

• Data standardisation

  GAPS :

  There is no standardisation for phenotypic data, except for the tests required by regulators to demonstrate drug or vaccine efficacies, which rely on in vivo testing.

• Climate change

• Role of disease control for climate adaptation

  The impact of climate on coccidiosis has not been evaluated in any detail although there is some evidence that climactic conditions may contribute to differential rates of genetic exchange between parasites (presumably because of different rates of survival/opportunities for co-infections and recombination). However, since coccidia are present world-wide, the impact of climate change may have relatively low impact.

  GAPS :

  Stresses of extreme weather, especially heat stress, will have impacts on productivity; if food intake is reduced due to stress, control of coccidiosis by in-feed anticoccidial drugs will be compromised. The importance of subclinical coccidiosis is likely to be exacerbated when birds are stressed.

• Effect of disease (control) on resource use

  Increased incidence will cause costs of poultry production to rise and may decrease affordability or availability of meat and eggs. Coccidiosis increases feed conversion ratios, which means more fodder is needed to produce the same outputs of meat or eggs.

• Effect of disease (control) on emissions and pollution (greenhouse gases, phosphate, nitrate, …)

  Coccidiosis causes diarrhoea and wet litter leading to increased ammonia production which impacts on bird health and welfare as well as increased emissions.

• Preparedness

• Syndromic surveillance

  Coccidiosis is well recognised and accounted for by farmers and veterinarians, but subclinical disease and its impact on productivity is under recognised and there is no tool for detection.

• Diagnostic platforms

  No high throughput methods are available commercially.

• Mathematical modelling

  There has been some academic work on modelling of disease transmission based on both experimental and field studies. Whilst there is good knowledge of some parasite parameters (eg prepatent times, replicative potential, survival in the environment), factors that influence outcomes such as host genetics, infectious load, age at first infection, presence of co-infections, nutritional status are not fully understood or accurately quantified.

  GAPS :

  Modelling the impact of parasite drug resistance, or the application of sub-optimal vaccines on disease transmission and impact on productivity is desirable and requires acquisition and analysis of production data.

• Intervention platforms

  Prophylaxis to control coccidiosis is always necessary to raise chickens commercially without encountering significant losses.

• Communication strategies

  Coccidiosis is of primary concern to the industry globally; in high income countries communication on control is well organised. In low income countries, information and knowledge of best practice is variable.

Main critical gaps

• 1. Mechanisms of drug resistance are not known in any detail; if these were elucidated, rapid tests for resistance would be useful for surveillance/screening of isolates.
  2. Despite a massive increase in good quality sequence data, there is little investment in identifying and characterising potential novel drug targets for both prophylaxis and for treatment of outbreaks.
  3. We need diagnostics tests or biomarkers that can distinguish between disease-free carrier status and subclinical coccidiosis.
  4. There are no general guidelines or tools for diagnosis of the newly identified species E. lata, E. nagambie and E. zaria (formerly OTUs).
  5. Methods to increase production of current vaccines, for example, in vitro culture methods for species of Eimeria, would potentiate greater dissemination and use of vaccines.
  6. Improved understanding of the mechanisms of protective immunity would support further development of vaccines.
  7. Better understanding of the basis of species-specificity and lack of cross-immunity between species of Eimeria could support generation of vaccines that protect against multiple species of parasite.
  8. Vaccines that can be used effectively in broilers and which can compete on price with drugs would support reduction in subclinical coccidiosis.
  9. Identification of additional vaccine antigens from all important species, and improvements in delivery methods and adjuvants are needed.
  10. Collaborations between academic researchers, poultry producers and veterinary pharma are needed both to support specific projects and to share knowledge/evidence on the effectiveness of interventions and the impact of coccidiosis.

Conclusion

• Coccidiosis remains a major problem for kept poultry around the world and infections must be controlled to avoid clinical disease which left unchecked cause high levels of morbidity and mortality, and to avoid subclinical disease which impacts on bird welfare and productivity.

  There are significant gaps in our knowledge of how precisely different species of Eimeria parasites interact with the chicken, their tropism for different parts of the gut, how they induce pathology and how the host mounts a protective and highly species-specific immune response that protects against reinfection. We also lack detailed understanding of the mechanisms by which parasites rapidly develop resistance to anticoccidial drugs, and there is only little understanding of parasite epidemiology and population genetics. From a small number of studies, we know that field isolates display high levels of genetic polymorphism and that in some cases this results in antigenic variation that contributes to evasion of vaccine-induced immunity. Since the last DISCONTOOLS evaluation, the isolation and characterisation of parasites representing three previously described ‘operational taxonomic units, (OTUs)’ has shown convincingly that these are new independent species of Eimeria of the chicken: E. lata (OTU-X), E. nagambie (OTU-Y) and E. zaria (OTU-Z). All three of these are common across the southern hemisphere and have also been detected in North America and southern Europe but so far their contribution to coccidiosis in the field is not understood. Importantly, whilst these new species may be controlled by anticoccidial drugs, they are not controlled by the current live vaccines.

  Current tools for diagnosis are insufficient especially when considering the global stage; classical methods are highly informative but not applicable to mass testing, whereas molecular tools, which are available in specialised settings, can identify and quantify infection but do not evaluate disease intensity (ie pathology). There are also no diagnostic tests that can identify drug resistance, differentiate between strains (including DIVA), or differentiate between animals with inapparent infections (coccidiasis) and subclinical coccidiosis.

  There are a range of pharmaceutical tools available for control or treatment of coccidiosis, although significantly fewer than in previous times due to companies deciding not to re-register compounds due to costs. For the moment, it is broadly accepted within Europe that in-feed anticoccidials remain an essential tool to control coccidiosis in fast-growing broilers, and in turkeys, however the debate continues and there is a strong desire in the veterinary community to ensure that use of coccidiostats is monitored effectively. It is notable that there is little investment in the development of new effective drugs, despite the recent availability of high-quality molecular sequence databases.

  Live vaccines have been available for decades and make important contributions to control of coccidiosis, especially in the breeder and layer sectors where they are widely deployed. In the USA, where retailer pressure is driving down the use of anticoccidial drugs in meat birds, live non-attenuated vaccines are used in around 40% of broiler production despite the impact that these vaccines can have on feed conversion and weight gain of these short-lived birds due to subclinical coccidiosis (caused by the re-cycling of fully virulent parasites). Within the EU where only live-attenuated vaccines are licenced for use and these are must be produced in SPF chickens, the relative cost of vaccines remains high compared to anticoccidial drugs and their use in broilers is much more limited. The goal of producing novel vaccines using molecular technologies (eg recombinant subunit, nucleic acid or live-vector expression) remains highly desirable, especially if such approaches could offer cross-protection against several Eimeria species and induce robust protection across a flock in less than three weeks.

Sources of information

• Expert group composition

  Fiona Tomley, Professor of Experimental Parasitology, Royal Veterinary College, London, UK – [Leader];

  Damer Blake, Professor of Parasite Genetics, Royal Veterinary College, London, UK;

  Marc Pagès Bosch, Senior Manager R&D Biologicals, Hipra, Girona, Spain;

  Jean-Michel Répérant, Senior researcher, Laboratoire de Ploufragan-Plouzané-Niort, ANSES, Ploufragan, France ;

  Monita Vereecken, DVM, Technical Manager Europe, Huvepharma, Antwerp, Belgium ;

  Vladimir Vrba, Process Innovation Manager, Eimeria Pty Ltd, Attwood, Victoria, Australia.

• Date of submission by expert group

  6 March 2023

• References

  There is a large literature available and the following review articles and recent original research papers will provide the reader with some useful starting material.

  Blake DP, Knox J, Dehaeck B, Huntington B, Rathinam T, Ravipati V, Ayoade S, Gilbert W, Adebambo AO, Jatau ID, Raman M, Parker D, Rushton J, Tomley FM. Re-calculating the cost of coccidiosis in chickens. Vet Res. 2020 Sep 14;51(1):115. doi: 10.1186/s13567-020-00837-2. PMID: 32928271; PMCID: PMC7488756.https://pubmed.ncbi.nlm.nih.gov/32928271/

  Blake DP, Marugan-Hernandez V, Tomley FM. Spotlight on avian pathology: Eimeria and the disease coccidiosis. Avian Pathol. 2021 Apr 20:1-5. doi: 10.1080/03079457.2021.1912288.https://www.tandfonline.com/doi/full/10.1080/03079457.2021.1912288

  Blake DP, Vrba V, Xia D, Jatau ID, Spiro S, Nolan MJ, Underwood G, Tomley FM. Genetic and biological characterisation of three cryptic Eimeria operational taxonomic units that infect chickens (Gallus gallus domesticus). Int J Parasitol. 2021 Jul;51(8):621-634. doi: 10.1016/j.ijpara.2020.12.004https://scholar.google.it/scholar?q=.+doi:+10.1016/j.ijpara.2020.12.004&hl=nl&as_sdt=0&as_vis=1&oi=scholart

  Chapman HD (2008). Coccidiosis in the turkey. Avian Pathology 37: 205-223.

  Chapman HD, Rathinam T. Focused review: The role of drug combinations for the control of coccidiosis in commercially reared chickens. Int J Parasitol Drugs Drug Resist. 2022 Apr; 18:32-42. doi: 10.1016/j.ijpddr.2022.01.001. https://pubmed.ncbi.nlm.nih.gov/35066424/

  Chapman HD, Roberts B, Shirley MW, Williams RB (2005). Guidelines for evaluating the efficacy and safety of live anticoccidial vaccines and obtaining apporoval for their use in chickens and turkeys. Avian Path. 34: 279-290.

  Feix AS, Cruz-Bustos T, Ruttkowski B, Joachim A. In vitro cultivation methods for coccidian parasite research. Int J Parasitol. 2022 Nov 15: S0020-7519(22)00153-9. doi: 10.1016/j.ijpara.2022.10.002https://pubmed.ncbi.nlm.nih.gov/36400306/

  Gilbert W, Bellet C, Blake DP, Tomley FM, Rushton J. Revisiting the Economic Impacts of Eimeria and Its Control in European Intensive Broiler Systems with a Recursive Modelling Approach. Front Vet Sci. 2020 Nov 5; 7:558182. doi: 10.3389/fvets.2020.558182.https://www.frontiersin.org/articles/10.3389/fvets.2020.558182/full

  Martins RR, Silva LJG, Pereira AMPT, Esteves A, Duarte SC, Pena A. Coccidiostats and Poultry: A Comprehensive Review and Current Legislation. Foods. 2022 Sep 7;11(18):2738. doi: 10.3390/foods11182738https://www.mdpi.com/2304-8158/11/18/2738

  Vereecken M, Dehaeck B, Rathinam T, Schelstraete W, De Gussem K, Chapman HD. Restoration of the sensitivity of Eimeria acervulina to anticoccidial drugs in the chicken following use of a live coccidiosis vaccine. Vet Parasitol. 2021 Apr; 292:109416. doi: 10.1016/j.vetpar.2021.109416.https://www.sciencedirect.com/science/article/abs/pii/S0304401721000765

  Vrba V, Pakandl M. Host specificity of turkey and chicken Eimeria: controlled cross-transmission studies and a phylogenetic view. Vet Parasitol. 2015 Mar 15;208(3-4):118-24. doi: 10.1016/j.vetpar.2015.01.017.https://pubmed.ncbi.nlm.nih.gov/25660426/

  Wang S, Suo X. Still naïve or primed: Anticoccidial vaccines call for memory. Exp Parasitol. 2020 Sep;216:107945. doi: 10.1016/j.exppara.2020.107945.https://www.sciencedirect.com/science/article/pii/S0014489420300308

  Williams RB (2005). Intercurrent coccidiosis and necrotic enteritis of chickens: rational, integrated disease management by maintenance of gut integrity. Avian Pathology 34: 159-180.

  Zaheer T, Abbas RZ, Imran M, Abbas A, Butt A, Aslam S, Ahmad J. Vaccines against chicken coccidiosis with particular reference to previous decade: progress, challenges, and opportunities. Parasitol Res. 2022 Oct;121(10):2749-2763. doi: 10.1007/s00436-022-07612-6.https://pubmed.ncbi.nlm.nih.gov/35925452/