The study aimed to monitor parasite and host gene expression during the early stages of Eimeria tenella infection of chicken cells using dual RNA-Seq analysis. For this, we used chicken macrophage-like cell line HD11 cultures infected in vitro with purified E. tenella sporozoites. Cultures were harvested between 2 and 72 h post-infection and mRNA was extracted and sequenced. Dual RNA-Seq analysis showed clear patterns of altered expression for both parasite and host genes during infection. For example, genes in the chicken immune system showed upregulation early (2–4 h), a strong downregulation of genes across the immune system at 24 h and a repetition of early patterns at 72 h, indicating that invasion by a second generation of parasites was occurring. The observed downregulation may be due to immune self-regulation or to immune evasive mechanisms exerted by E. tenella. Results also suggested pathogen recognition receptors involved in E. tenella innate recognition, MRC2, TLR15 and NLRC5 and showed distinct chemokine and cytokine induction patterns. Moreover, the expression of several functional categories of Eimeria genes, such as rhoptry kinase genes and microneme genes, were also examined, showing distinctive differences which were expressed in sporozoites and merozoites.

Coccidiosis is an antagonistic poultry disease which negatively impacts animal welfare and productivity. The disease is caused by an obligate, intracellular protozoon known as Eimeria. Several Eimeria species known to infect chickens have been well documented. However, recent studies have elucidated the emergence of three novel genetic variants or operational taxonomic units (OTUs). The discovery of OTUx, OTUy and OTUz complicates the identification and diagnosis of coccidiosis. OTUs are clusters of unknown or uncultivated organisms that are grouped according to a similarity in DNA sequence to a set of specific gene markers. OTUs have been reported in the Earth's Southern Hemisphere, including Australia, Venezuela, India, Zambia, Uganda, Tanzania, China and Ghana. Elucidating their impact on the poultry industry is fundamental in preventing anticoccidial resistance and to access the potential of OTUs as vaccine candidates to provide cross-protection against similar Eimeria species. The identification of OTUs further decreases the risk of false negative coccidial diagnosis. Therefore, this article reviews the importance and risk imposed by OTUs, coupled with their prevalence and geographical distribution in chickens globally.

Coccidial infections reduce fat-soluble vitamin status and bone mineralisation in broiler chickens. We hypothesised that broilers infected with Eimeria maxima would benefit from increased dietary supplementation with vitamin D (vitD) or with 25-hydroxycholecalciferol (25(OH)D3 or 25D3). Broilers were assigned to diets with low (L) or commercial (M) vitD levels (25 v. 100 μg/kg) supplemented as cholecalciferol (D3) or 25D3. At day 11 of age, birds were inoculated with water or 7000 E. maxima oocysts. Pen performance was calculated over the early (days 1–6), acute (days 7–10) and recovery periods (days 11–14) post-infection (pi). At the end of each period, six birds per treatment were dissected to assess long bone mineralisation, plasma levels of 25D3, Ca and P, and intestinal histomorphometry. Parasite replication and transcription of cytokines IL-10 and interferon-γ (IFN-γ) were assessed at day 6 pi using quantitative PCR. Performance, bone mineralisation and plasma 25D3 levels were significantly reduced during infection (P < 0·05). M diets or diets with 25D3 raised plasma 25D3, improved performance and mineralisation (P < 0·05). Offering L diets compromised feed efficiency pi, reduced femur breaking strength and plasma P levels at day 10 pi in infected birds (P < 0·05). Contrastingly, offering M diets or diets with 25D3 resulted in higher parasite loads (P < 0·001) and reduced jejunal villi length at day 10 pi (P < 0·01), with no effect on IL-10 or IFN-γ transcription. Diets with M levels or 25D3 improved performance and mineralisation, irrespective of infection, while M levels further improved feed efficiency and mineralisation in the presence of coccidiosis.

Infection with Eimeria sp. results in the activation of multiple facets of the host immune system; the use of phytogenics can modulate the inflammatory response and improve the performance of the challenged animal. The aim of this study was to evaluate the effect of a commercial blend of cashew nut shell liquid (CNSL) and castor oil on the immune response of broilers challenged with coccidiosis. A total of 864 one-day-old male chicks (Cobb 500) were randomly distributed into six treatment groups (8 pens/treatment and 18 chicks/pen) in a three-by-two factorial design with three additives: control (non-additive), 100 ppm of monensin or 0.15% CNSL–castor oil. Challenge status was determined twice at 14 days of age. Unchallenged birds were inoculated by gavage with oocysts sporulated with Eimeria tenella, Eimeria acervulina and Eimeria maxima. Although the positive control (non-additive and challenged) and CNSL–castor oil treatment groups exhibited similar variation in weight gain (ΔBWG) compared to unchallenged birds fed without additives, the variation observed in birds fed diets containing CNSL–castor oil was associated with a higher maintenance requirement and not feed efficiency. In the second week after infection, ΔBWG of the CNSL–castor oil treatment group did not significantly change compared to the other treatment groups. At days 7 and 14 post-challenge, there was a higher excretion of oocysts in the control group, whereas the CNSL–castor oil and monensin groups did not differ. The CNSL–castor oil group exhibited increased gene expression of interferon (IFN), interleukin 6 (IL-6) and tumor necrosis factor (TNF), while the control group exhibited increased expression of cyclooxygenase (COX) and IL-1. The heterophils/lymphocyte ratio was low for the monensin treatment group. The unchallenged birds that received monensin treatment presented higher gene expression of IFN, COX and IL-1 compared to the other treatments, while the CNSL–castor oil group exhibited reduced gene expression, except for TNF. The commercial blend of cashew nut liquid and castor oil modulated the inflammatory response against Eimeria spp. In the absence of the parasite, there was no stimulation of genes involved in the inflammatory response, demonstrating that the blend is an effective tool in specifically modulating the immune system of birds afflicted with coccidiosis.

Necrotic enteritis (NE) has become one of the most important diseases of modern global poultry production, with an estimated cost of around US$6 billion per annum in lost production and control strategies. The rise in prominence of NE is attributed to the prohibition of antibiotics as growth promoters (AGP) or the voluntary implementation of ‘drug-free’ broiler production programmes. Pathogenic strains of Clostridium perfringens are responsible for NE, with those expressing the NetB toxin a definitive cause in disease models. C. perfringens are normal inhabitants of the gastrointestinal tract (GIT) but these are typically non-pathogenic strains. When intestinal health is compromised, the prevailing conditions allow the establishment and proliferation of pathogenic, toxin-secreting strains of C. perfringens. The toxin(s) damages the intestinal epithelium and causes disease. Certain dietary-related factors are recognised as predisposing poultry to NE. This review will focus on the key initiators of NE and will outline the most appropriate strategies to counteract these predisposing factors and prevent NE. The continual push, globally, for poultry production programmes with less antibiotic use will sustain NE as an important and costly poultry disease that requires dietary intervention.

For this study, threonine (Thr) deficiency was hypothesised to exacerbate the intestinal damage induced by feed withdrawal with coccidial infection because of its high obligatory requirement by the gut; two dietary Thr treatments (0·49 and 0·90 %) were applied to chicks from 0 to 21 d of age. At 13 d of age, feed was withdrawn for 24 h from one-half of birds of each dietary treatment with subsequent gavage of a 25× dose of coccidial vaccine. Overall, there were four treatments with eight replicate cages per treatment. Under combined challenge, birds fed the Thr-deficient diet had 38 % lower 13–21-d body weight gain (P≤0·05) compared with birds fed the Thr-control diet. At 21 d, the challenged group fed low Thr had higher number of oocysts (+40 %, P=0·03) and lower crypt depth (−31 %, P<0·01). In addition, birds fed the low-Thr diet had higher gut permeability as measured after 2 h of administration of fluorescein isothiocyanate-dextran (3–5 kDa, P<0·01), which may be attributed to decreased IgA production (P=0·03) in the ileum. In caecal tonsils, the challenged group fed low Thr had lower CD3:Bu-1 ratio (P≤0·05), along with a tendency for lower CCR9 mRNA expression in birds fed the low-Thr diet (P=0·10). In addition, Thr deficiency tended to increase IL-10 mRNA expression regardless of infection (P=0·06), but did not change interferon-γ mRNA expression upon coccidial infection (P>0·05). Overall, Thr deficiency worsened the detrimental effects of combined feed withdrawal and coccidial infection on growth performance and oocyst shedding by impairing intestinal morphology, barrier function, lymphocyte profiles and their cytokine expressions.

The poultry industry is currently facing a serious problem of controlling coccidiosis, owing to the development of drug resistance against commonly available anticoccidials. Furthermore, an increasing demand in the consumers for drug residue free poultry and poultry products has led to the development of alternative strategies for the treatment and control of avian coccidiosis. In response to the invasion of Eimeria species in coccidiosis, oxidative stress is created by host cellular response which imparts pathology to the host tissue besides damaging the parasite. Hence, in order to alleviate the damage caused by oxidative stress during coccidiosis, the use of essential oils (EOs) rich in antioxidant compounds is being considered as an appealing approach. However, results are very divergent and often not as satisfactory as expected. Essential oils, as natural products, obtained from aromatic plants have the potential to serve as an alternate to anticoccidials. The present work aims to review the current state of knowledge, informative collection of results obtained over the years and to attain a critical decision in aspects of the use of EOs as anticoccidials.

This study evaluated the effect of an enzyme blend (xylanase, amylase and protease; XAP) in combination with a direct fed microbial (DFM) containing three strains of Bacillus spp. on intestinal histology, immune response and performance of broilers. Four dietary treatments were tested in a 2 × 2 factorial trial, including two levels of challenge (without or with coccidial infection), two levels of feed additive (with or without XAP and DFM). Diets were fed ad libitum to male Cobb500 broilers in mash feeds from 1–21 days of age, with eight replicate pens per treatment within brooder-batteries with raised wire floors and built up litter, housing six birds per pen. A mild challenge was introduced by oral gavage at day five to the challenged birds, using a six-fold concentration of coccidial vaccine. A high fibre basal diet formulated with rye and wheat middlings was used to further increase the challenge. Body weight and feed intake were measured and feed conversion ratio (FCR) was calculated during starter (1–12 d), grower (12–21 d) and overall 1–21 days. Intestinal morphology and immune response parameters were measured on day 12 and 21. Compared to the unchallenged groups, the coccidial challenge reduced (P < 0.05) body weight gain (BWG), increased FCR, reduced villus height and increased crypt depth. The challenged birds had increased pro-inflammatory cytokines (IL-6, IL-1β; P < 0.05) in the intestine as well as higher levels of acute phase proteins (APP, haemopexin and α−1-acid glycoprotein) in the plasma and circulating heterophils. XAP + DFM supplementation improved BWG, reduced FCR and increased energy efficiency compared to the non-supplemented groups. The combination of XAP and DFM reduced inflammatory responses such as APP compared to the challenged control group and maintained performance to a comparable level seen in the unchallenged control. The data indicate that XAP enzymes in combination with Bacillus-based DFM may reduce the damage and performance losses induced by coccidial challenge.

Coccidiosis is well known as an expensive, parasitic disease for the poultry industry worldwide. The disease causes real economic losses by causing poor growth and feed efficiency in broilers even leading to high mortality. Consequently, large amounts of money are being spent on vaccination and inclusion of anticoccidial drugs into diets. In recent years, development of resistance to coccidiostats, elevated costs of systematic vaccination and increasing consumer demand for ′natural′ food products has fuelled the development of natural, plant-based alternatives for coccidial control in poultry farming. The anti-coccidial properties of numerous natural products such as Ageratum conyzoides extract (billy goat weed), green tea, maslinic acid, extracts of Polygonum bistorta (Anjbar) and Agele marmelos (Bael), Artemisia sieberi, Artemisia absinthium, the leaves of Neem (Azadirachta indica), Artemisia annua and Aloa vera-based supplements have been reported. This article summarises the experimental knowledge relating to the efficacy, possible modes of action and different aspects of application of botanical supplements as feed additives for the treatment of poultry diseases, especially coccidiosis.

The world poultry industry is under great stress due to parasitic diseases; coccidiosis being one which is responsible for substantial economic losses worldwide. In 1948 the first research paper was published about the treatment of coccidiosis with sulphaquinoxaline. After six decades, researcher's attention has focused on DNA vaccination, especially as certain anticoccidials have failed due to drug resistance and residues. Thus far vaccination is partially successful but is accompanied by disadvantages: e.g. instability, inferiority control, cost-effectiveness, and inefficiency in opposition to a large number of coccidian strains which are prevalent in different geographical areas. Due to developments whereby genetically engineered DNA can be administered in vaccine form to provoke cellular and humoral immune responses; there has been huge development in the practical application of this field. In the last decade a number of DNA vaccines employing different strategies have been tested to produce appropriate immune responses against coccidiosis. The DNA fragments taken from all the four important species, E. tenella, E. necatrix, E. maxima and E. acervulina were able to provoke appropriate immune responses against challenging infections with homologous species; however, most of them were not able to provoke a response with heterologous infection. The shared DNA antigen in two different species of Eimeria; E. tenella and E. acervulina was able to produce sufficient immune responses, not only against these species but also against E. necatrix, but not against E. maxima. E. maximum is the biggest and most complex of all the seven species and it has come ahead as a challenge for DNA vaccine researchers.

There has been growing interest in the development of natural methods of coccidiosis prophylaxis. Vaccination against coccidiosis with live oocysts is regarded as effective in the control of avian coccidiosis, but in some conditions, such as poor sanitary management or incorrect application of the vaccine, may lead to poorer performance, measured as reduced weight gain and feed efficiency, in comparison with prophylactically medicated birds. Such reports are the reason for widespread reluctance to use anticoccidial vaccine in broilers. This paper reviews broiler studies in which nutritional methods as performance and/or immune-supporting factor with regard to anticoccidial vaccination were investigated. The results of some of the studies presented in the article indicate that feed additives with immunostimulating properties, along with proper protein levels in the diet, may have a beneficial effect in cocci-vaccinated broilers.

The objective of the present study was to evaluate the anticoccidial effect of different concentrations of the herbal complex of 4 plants (leaves of Azadirachta indica and Nicotiana tabacum, flowers of Calotropis procera and seeds of Trachyspermum ammi) in broiler chickens in comparison with amprolium anticoccidial. Three concentrations (2 g, 4 g and 6 g) of herbal complex were given to the experimental groups once a day and amprolium (at the dose rate of 125 ppm) was given orally in drinking water from the 14th to the 21st days of age. One group was kept as infected, non-medicated control and one as non-infected, non-medicated control. All groups were inoculated orally with 75 000 sporulated oocysts on the 14th day of age except the non-infected, non-medicated control. Among herbal complex medicated groups, the maximum anticoccidial effect was seen in the group medicated with 6 g herbal complex followed by 4 g and 2 g herbal complex medicated groups. Treatment with 6 g of the herbal complex significantly reduced the negative performance and pathogenic effects associated with Eimeria tenella challenge at a level that was comparable with amprolium when using a largely susceptible recent field isolate. In summary, concentration-dependent anticoccidial activity of the studied herbal complex suggests its use as an alternative anticoccidial agent to chemotherapeutic drugs for Eimeria tenella control.

Although oocyst morphology was always considered as a reliable parameter for coccidian species discrimination we describe strain variation of turkey coccidia, Eimeria adenoeides, which remarkably exceeds the variation observed in any other Eimeria species. Two strains have been isolated – the first strain maintains the typical oocyst morphology attributed to this species – large and ellipsoidal – while the second strain has small and ovoid oocysts, never described before for this species. Other biological parameters including pathogenicity were found to be similar. Cross-protection between these 2 strains in 2 immunization and challenge experiments was confirmed. Sequencing and analysis of 18S and ITS1 ribosomal DNA revealed a close relationship according to 18S and a relatively distant relationship according to ITS1. Analysis of 18S and ITS1 sequences from commercial turkey coccidiosis vaccines Immucox®-T and Coccivac®-T revealed that each vaccine contains a different strain of E. adenoeides and that these strains have 18S and ITS1 sequences homologous to the sequences of the strains we have isolated and described. These findings show that diagnostics of turkey coccidia according to oocyst morphology have to be carried out with caution or abolished entirely. Novel PCR-based molecular tools will be necessary for fast and reliable species discrimination.

The effects of cinnamaldehyde (CINN) on in vitro parameters of immunity and in vivo protection against avian coccidiosis were evaluated. In vitro stimulation of chicken spleen lymphocytes with CINN (25–400 ng/ml) induced greater cell proliferation compared with the medium control (P < 0·001). CINN activated cultured macrophages to produce higher levels of NO at 1·2–5·0 μg/ml (P < 0·001), inhibited the growth of chicken tumour cells at 0·6–2·5 μg/ml (P < 0·001) and reduced the viability of Eimeriatenella parasites at 10 and 100 μg/ml (P < 0·05 and P < 0·001, respectively), compared with media controls. In chickens fed a diet supplemented with CINN at 14·4 mg/kg, the levels of IL-1β, IL-6, IL-15 and interferon-γ transcripts in intestinal lymphocytes were 2- to 47-fold higher (P < 0·001) compared with chickens given a non-supplemented diet. To determine the effect of CINN diets on avian coccidiosis, chickens were fed diets supplemented with CINN at 14·4 mg/kg (E. maxima or E. tenella) or 125 mg/kg (E. acervulina) from hatch for 24 d, and orally infected with 2·0 × 104 sporulated oocysts at age 14 d. CINN-fed chickens showed 16·5 and 41·6 % increased body-weight gains between 0–9 d post-infection (DPI) with E. acervulina or E. maxima, reduced E. acervulina oocyst shedding between 5–9 DPI and increased E. tenella-stimulated parasite antibody responses at 9 DPI compared with controls.

Limitations with current chemotherapeutic and vaccinal control of coccidiosis caused by Eimeria species continue to prompt development of novel controls, including the identification of new drug targets. Glucose-6-phosphate isomerase (G6-PI) has been proposed as a valid drug target for many protozoa, although polymorphism revealed by electrophoretic enzyme mobility has raised doubts for Eimeria. In this study we identified and sequenced the Eimeria tenella G6-PI orthologue (EtG6-PI) from the reference Houghton strain and confirmed its position within the prevailing taxonomic hierarchy, branching with the Apicomplexa and Plantae, distinct from the Animalia including the host, Gallus gallus. Comparison of the deduced 1647 bp EtG6-PI coding sequence with the 9016 bp genomic locus revealed 15 exons, all of which obey the intron-AG-/exon/-GT-intron splicing rule. Comparison with the Weybridge and Wisconsin strains revealed the presence of 33 single nucleotide polymorphisms (SNPs) and 14 insertion/deletion sites. Three SNPs were exonic and all yielded non-synonymous substitutions. Preliminary structural predictions suggest little association between the coding SNPs and key G6-PI catalytic residues or residues thought to be involved in the coordination of the G6-PI's substrate phosphate group. Thus, the significant polymorphism from its host orthologue and minimal intra-specific polymorphism suggest G6-PI remains a valid anti-coccidial drug target.

The present review focuses on potential nutritional and physiological functions of betaine in relation to health, performance and carcass characteristics in poultry. Due to its zwitterionic structure, betaine has osmoprotective properties that aid in protecting intestinal cells and thus counteracting performance losses during heat stress and coccidiosis. Hence, betaine might be an appropriate agent in the prevention of heat stress in livestock. In addition, during coccidiosis, the efficacy of betaine appears to be related to the Eimeria species, type of coccidiostat used and the severity of infection. As this compound has methyl donor properties, betaine is involved in methylation reactions in the organism and may therefore partly substitute other methyl group donors such as methionine and choline. Due to its methyl donor and osmoprotectant functions, recent research has shown that betaine improves nutrient digestibility, growth performance and feed conversion in broiler chicks, turkeys and meat ducks. Other potential benefits of the inclusion of betaine in poultry diets are lowering carcass fat content and increasing breast meat yield which may be of interest to satisfy consumer needs.

An experiment was conducted to determine the rate and maximum percentage of sporulation of Eimeria acervulina oocysts at various environmental conditions relating to temperature (21 versus 33 °C) and relative humidity (RH) (40 versus 80%). Measurements were made during 44 h after excretion of oocysts in 3 substrates: dry litter, clammy litter and pure faeces respectively. Maximum sporulation percentage in both dry (22·6%) and clammy litter (19·5%) was higher (P < 0·005) than in pure faeces (11·6%). Neither temperature nor RH had a significant influence on percentage of oocysts that sporulated. Under these simulated practical conditions approximately 25% of all oocysts sporulated, whereas sporulation under optimal conditions (29 °C, aeration, 2% K2Cr2O7) showed a higher (68%) sporulation ability of oocysts. At 33 °C sporulation proceeded at a faster pace than at 21 °C (P < 0·005). With respect to RH and substrate, once sporulation started, the rate of increase to maximum percentage was not different. Time of onset of sporulation was influenced by temperature (P < 0·0001) and RH (P < 0·001). Time of onset occurred 15 h later at 21 °C compared with 33 °C and 5 h later at 40% RH compared with 80%. Also, an interaction effect (P < 0·01) was found with effect of RH being stronger at 21 °C compared with 33 °C. It was concluded that the most important aspect in the epidemiology of E. acervulina during a flock cycle is the time of onset of sporulation with temperature being the most important factor.

The pattern of oocyst production of 8 inbred lines of chickens was compared for each of the 7 species of Eimeria which infect this host. Both the overall numbers and the pattern of oocyst production differed in the inbred lines, but there was no evidence of prolonged cycling of schizogenic developmental stages. Comparison of the numbers of oocysts produced by the different lines indicates that there may be common genetic factors affecting susceptibility to 6 of the 7 species. Surprisingly there appears to be an inverse relationship between susceptibility to E. tenella and susceptibility to the other species: lines which produced most oocysts of E. tenella produced least oocysts of the other species and vice-versa.

A simulation model for effects of Eimeria acervulina infection on technical and economic characteristics in broiler production is presented. The model describes development over time of the growth depression, feed intake reduction, and decrease in feed efficiency associated with infection. The model also shows a phase of compensatory growth during which earlier negative effects are counterbalanced, at least partly. Major assumptions made were: infection with E. acervulina occurs in each flock; production is affected in each flock; compensatory growth takes place because immunity develops and cell regeneration occurs. The results show that the pattern of development of the production characteristics during a flock cycle depends on the initial contamination level. Both a high and low initial contamination level results in a lower average daily gain, a worse feed to gain ratio, and a reduced net revenue compared to an intermediate contamination level.

A simulation model for the population dynamics of Eimeria acervulina infection in broilers is presented. The model describes the development of the numbers of parasites in the various life-stages during the growing period of broilers and the empty house period between grow-outs. The model includes assumptions with respect to development of immunity to E. acervulina infection and effects of application of anticoccidial drugs. The model consists of a set of difference equations that are solved numerically at 1 h intervals. Under constant conditions, an equilibrium level was reached after a few grow-outs during which infection always peaked around the 21st day in the growing period. Within a growing period, infection peaked earlier (later) than the 21st day in case initial numbers of sporulated oocysts were higher (lower) than the equilibrium number.