Avian Coccidiosis Research Articles

Precision intestinal nutrition: knowledge and gaps regarding the role of amino acids during an enteric challenge

Poultry nutritionists continually strive for more “precision” nutritional programs that provide the exact balance of nutrients that maximize broiler growth performance without economically and environmentally costly excesses. Many factors affect the precise amount and balance of nutrients needed by the broiler, including genetics, age, sex, and environment. Furthermore, broilers in intensive rearing environments will almost always be subjected to some degree of enteric stress that can alter nutrient needs. Exposure to enteric pathogens such as Eimeria spp., the intestinal parasites that cause avian coccidiosis, induces physical damage to the intestinal epithelium and activates immune responses, ultimately resulting in the repartitioning of amino acids (AA) in response to these prioritized demands. Even without any pathogenic challenge, the intestine has an already high demand for many AA, with 30 to 100% of dietary AA extracted during first pass intestinal metabolism. In many cases, increasing dietary protein from intact proteins has been shown to be a viable option to ameliorate impaired AA digestion and absorption and heightened need for certain AA of birds under an enteric stress. However, increasing dietary protein often results in concomitant increases in indigestible protein and carbohydrates that can stimulate the overgrowth of pathogenic bacteria (i.e., Clostridium perfringens). Alternative options to increase dietary AA levels are to increase all feed-grade, free AA (e.g., Met, Lys, Thr, Val), or specific individual feed-grade AA. Therefore, the objectives of this paper are to discuss precision nutrition, the dietary AA demands of the intestine, consequences of coccidiosis on AA needs of the intestine, and formulation approaches to meet these altered needs. In summary, increased dietary protein met by intact proteins has consistently demonstrated its benefits during an Eimeria spp. infection; however, to further the goal of precision nutritional programs, feeding higher levels of a specific AA to support desired functions such as intestinal recovery or immune function for birds experiencing an enteric stress still require further evaluation.

Eimeria maxima Rhomboid-like Protein 5 Provided Partial Protection against Homologous Challenge in Forms of Recombinant Protein and DNA Plasmid in Chickens.

Eimeria maxima (E. maxima) is one of the most prevalent species that causes chicken coccidiosis on chicken farms. During apicomplexan protozoa invasion, rhomboid-like proteins (ROMs) cleave microneme proteins (MICs), allowing the parasites to fully enter the host cells, which suggests that ROMs have the potential to be candidate antigens for the development of subunit or DNA vaccines against coccidiosis. In this study, a recombinant protein of E. maxima ROM5 (rEmROM5) was expressed and purified and was used as a subunit vaccine. The eukaryotic expression plasmid of pVAX–EmROM5 was constructed and was used as a DNA vaccine. Chickens who were two weeks old were vaccinated with the rEmROM5 and pVAX–EmROM5 vaccines twice, with a one-week interval separating the vaccination periods. The transcription and expression of pVAX–EmROM5 in the injected sites were detected through reverse transcription PCR (RT-PCR) and Western blot (WB) assays. The cellular and humoral immune responses that were induced by EmROM5 were determined by detecting the proportion of CD4+ and CD8+ T lymphocytes, the cytokine levels, and the serum antibody levels. Finally, vaccination-challenge trials were conducted to evaluate the protective efficacy of EmROM5 in forms of the recombinant protein (rEmROM5) and in the DNA plasmid (pVAX–EmROM5) separately. The results showed that rEmROM5 was about 53.64 kDa, which was well purified and recognized by the His-Tag Mouse Monoclonal antibody and the chicken serum against E. maxima separately. After vaccination, pVAX–EmROM5 was successfully transcribed and expressed in the injected sites of the chickens. Vaccination with rEmROM5 or pVAX–EmROM5 significantly promoted the proportion of CD4+/CD3+ and CD8+/CD3+ T lymphocytes, the mRNA levels of the cytokines IFN-γ, IL-2, IL-4, IL-17, TNF SF15, and IL-10, and specific IgG antibody levels compared to the control groups. The immunization also significantly reduced the weight loss, oocyst production, and intestinal lesions that are caused by E. maxima infection. The anticoccidial index (ACI)s of the vaccinated groups were beyond 160, showing moderate protection against E. maxima infection. In summary, EmROM5 was able to induce a robust immune response and effective protection against E. maxima in chickens in the form of both a recombinant protein and DNA plasmid. Hence, EmROM5 could be used as a candidate antigen for DNA vaccines and subunit vaccines against avian coccidiosis.

Molecular characterization and immune protection by cystathionine β-synthase from Eimeria tenella.

Eimeria tenella is an obligate intracellular apicomplexan parasite that causes avian coccidiosis and leads to severe economic losses in the global poultry industry. Cystathionine β-synthase (CBS) and cystathionine γ-lyase (CGL) act together to generate H2S in the reverse transsulfuration pathway. In this study, E.tenella Cystathionine β-synthase (EtCBS) was cloned using rapid amplification of cDNA 5'-ends (5'RACE) and characterized, and its immunoprotective effects were evaluated. The recombinant EtCBS protein (rEtCBS) was expressed and successfully recognized by anti-sporozoites (Spz) protein rabbit serum. EtCBS mRNA levels were highest in Spz by qPCR, and the protein expression levels were higher in unsporulated oocysts (UO) than in other stages by Western blot. Indirect immunofluorescence showed that EtCBS protein was found on the surface of Spz and second-generation merozoites (Mrz). The invasion inhibition assays showed that rabbit anti-rEtCBS polyclonal antibodies effectively inhibited parasite invasion host cells. Chickens immunized with rEtCBS protein showed prominently increased weight gains and decreased oocyst output compared to nonimmunized and infected control group. The results suggest that EtCBS could be a potential vaccine candidate against E.tenella.

Immunotherapy With Egg Yolk Eimeria sp.-Specific Immunoglobulins in SPF Leghorn Chicks Elicits Successful Protection Against Eimeria tenella Infection.

Avian coccidiosis is the first to most economically important parasite disease affecting poultry industries worldwide. Current prevention measures are largely based upon prophylactic chemotherapy supplemented by the application of live attenuated or wild-type parasite vaccines. However, the rising appearance of drug resistance, consumer's concern for antibiotics use in poultry production and higher manufacturing cost of live vaccines has driven to adopt new technologies aimed at increasing animal health and production efficiency. Supplementing chickens with egg yolk Eimeria sp.-specific immunoglobulins can be a viable alternative to avoid severe outbreaks of the disease. Twelve-week-old SPF White Leghorn chickens were experimentally infected with a large dose of E. tenella. During the prepatent period, the birds were supplemented by oral gavage with 60 or 120 mg/bird of hyperimmune egg yolk Eimeria species-specific immunoglobulins Y (Supracox®, SC) on a daily basis. The animals were euthanized 7 days post-infection (PI) and their passive immune protection was evaluated. Birds treated with 120 mg/bird of SC showed more viability, increased body weight gain (BWG), a normal hematocrit level (HCT), reduced oocyst output per gram of feces (OPG) or cecal tissue (OPGC), and fewer cecal lesions compared to the untreated infected (UI) control group. Birds supplemented with 60 mg/bird of SC did not show any significant difference on BWG, HCT, OPG, OPGC, and cecal lesion score when compared with the UI group. An ELISA test of the SC showed a weak cross-reactivity of IgY toward two asexual zoite stages of E. tenella. Western blot analysis of the sporozoite with SC showed few antigens barely recognized, while more stained bands were detected in the merozoite (≈82, ≈60, ≈54, ≈40, ≈38, ≈27.5, and ≈13 kDa). Oral immunotherapy using egg yolk polyclonal IgYs against Eimeria sp. represents an effective and natural resource against severe E. tenella infection favoring the gradual withdrawal of the anticoccidial drugs and antibiotics.

Anticoccidial potential of Ageratum conyzoides and its effect on Blood parameters of experimentally infected Broiler Chickens

Coccidiosis is an infectious parasitic disease of poultry which causes heavy economic losses to commercial poultry industry. Coccidiosis has been generally controlled by using different synthetic anticoccidial drugs but, due to development of resistance to these drugs this method is losing its effectiveness. Exploration of novel and alternative compounds against avian coccidiosis is need of time now a days. In this regard, medicinal plants can serve as substitute to these synthetic anticoccidials. Thus, to find out alternative novel agents, current research was designed to evaluate the anticoccidial potential of Ageratum conyzoides extract (ACE). For in vivo experiment, 105 broiler chicks were purchased and further divided into 7 sub-groups (15 birds in each group). At 7th day of experiment, Groups A, B and C were fed with plant extract at 100, 200 and 300 mg/kg respectively. Group D was supplemented with Vitamin-E while, Group E and F remained as infected medicated and infected un-medicated control groups and served with standard medicine (Baycox®) and Phosphate Buffer Saline (PBS) respectively. Moreover, Group G served as normal control group. At day 14th of experiment, all groups except rom Group G were orally infected with 60,000 sporulated oocysts of mixed Eimeria species. Anticoccidial potential of ACE was evaluated on the basis of lesion scores, fecal scores, oocyst scores and feed conversion ratio. Furthermore, the effect of ACE on the serum chemistry was also evaluated to check toxicity of plant extract if any. The results were compared with standard medicine (Baycox®). On the basis of results, ACE showed anticoccidial activity by reducing fecal, lesion and oocyst scores in infected chicks (P<0.05). ACE also improved FCR of infected chicks. Moreover, ACE exhibited positive effects on serum chemistry of broiler chickens (P<0.05).

Development of Garlic Oral Beads using Garlic Nanoparticles and Studies on their Protective Effects Against Avian Coccidiosis

In this paper, new oral beads were developed for poultry applications using garlic nanoparticles (NPs). The NPs were synthesized using water in oil micro-emulsion method and it was confirmed using energy dispersive X-ray spectroscopy (EDX), Fourier transform infrared spectroscopy (FTIR) and Raman spectroscopy. The biocompatibility was evaluated by using the chicken blood, chicken intestinal cell line and the results indicated that the particles are highly safe. The oral beads were formulated using hydroxyl propyl methyl cellulose phthalate as polymer protective layer and specifically dissolved in pH 6.5 and released the NPs at the intestine. To study their impacts, the chickens were fed with beads starting day 3 after hatching, and their weight was recorded. To study the anti-parasitic effect, the chickens were challenged with Eimeria species. The active treatments with oral beads effectively cure the intestinal damages triggered by the Eimeria species and improved the chicken weight. The prophylactic treatment 100% protects chicken against Eimeria infection and increased the body weight.

Genetic Diversity of Microneme Protein 2 and Surface Antigen 1 of Eimeria tenella.

Avian coccidiosis is a disease caused by members of the genus Eimeria. Huge economic losses incurred by the global poultry industry due to coccidiosis have increased the need for cost-effective and easily available recombinant vaccines. Microneme protein 2 (MIC2) and surface antigen 1 (SAG1) of E. tenella have been recognised as potential vaccine candidates. However, the genetic diversity of the antigens in field isolates, which affects vaccine efficacy, has yet to be largely investigated. Here, we analysed genetic diversity and natural selection of etmic2 and etsag1 in Korean E. tenella isolates. Both genes exhibited low levels of genetic diversity in Korean isolates. However, the two genes showed different patterns of nucleotide diversity and amino acid polymorphism involving the E. tenella isolates obtained from different countries including China and India. These results underscore the need to investigate the genetic diversity of the vaccine candidate antigens and warrant monitoring of genetic heterogeneity and evolutionary aspects of the genes in larger numbers of E. tenella field isolates from different geographical areas to design effective coccidial vaccines.

Relationship between Eimeria tenella associated-early clinical signs and molecular changes in the intestinal barrier function

The major clinical signs of coccidiosis in chickens due to Eimeria parasite are diarrhea and bloody feces. Previous studies showed that the impairment of the intestinal epithelial barrier and the elevation of the intestinal permeability are causes of clinical signs associated with coccidia challenges. Nevertheless, the information about molecular changes of the epithelial barrier at the early stage of the infection with a specific Eimeria species has not been mentioned. Hence, this study aims to elucidate the temporal relationships between epithelial barrier conditions and clinical signs in chickens infected with Eimeria tenella over the time from the earliest stages of infection.White Leghorn chickens were inoculated with 1 × 104 oocysts of E. tenella. Thereafter the chickens were monitored for their daily clinical signs through observation, and between 5 dpi to 10 dpi, feces were collected for oocysts counting. Chickens were then administrated with fluorescein isothiocyanate-dextran (FITC-d) for gastrointestinal permeability test and tissues were collected each day for histopathological observation and total RNA extraction. Finally, the mRNA expression levels of the tight and adherens junction genes and cytokine genes were evaluated using the quantitative real-time polymerase chain reaction (qRT-PCR).In this study, clinical signs such as diarrhea and bloody feces were observed concurrently from 3 to 8 dpi. Histopathology changes such as severe inflammation, hemorrhage, and epithelial desquamation were identified in the cecum specimens. The FITC-d level in the E. tenella-infected group was significantly higher than in the control group. In the infected group, the expression of claudin-2 gene was also upregulated, whereas the expressions of claudin-3 and E-cadherin genes were decreased as compared to the control group. These results implied that clinical signs of avian coccidiosis were associated with the intestinal barrier disruption via changes in expression levels of claudins and E-cadherin at the intestine.

Anticoccidial Activity of Berberine against Eimeria-Infected Chickens.

Avian coccidiosis has a major economic impact on the poultry industry, it is caused by 7 species of Eimeria, and has been primarily controlled using chemotherapeutic agents. Due to the emergence of drug-resistant strains, alternative control strategies are needed. We assessed anticoccidial effects of berberine-based diets in broiler chickens following oral infection with 5 Eimeria species (E. acervulina, E. maxima, E. tenella, E. mitis, and E. praecox). When 0.2% berberine, a concentration that does not affect weight gain, was added to the diet, the 4 groups infected with E. acervulina, E. tenella, E. mitis, or E. praecox showed significant reductions in fecal oocyst shedding (P<0.05) compared to their respective infected and untreated controls. In chickens treated 0.5% berberine instead of 0.2% and infected with E. maxima, fecal oocyst production was significantly reduced, but body weight deceased, indicating that berberine treatment was not useful for E. maxima infection. Taken together, these results illustrate the applicability of berberine for prophylactic use to control most Eimeria infections except E. maxima. Further studies on the mechanisms underlying the differences in anticoccidial susceptibility to berberine, particularly E. maxima, are remained.

Genome-Wide Expression Patterns of Rhoptry Kinases during the Eimeria tenella Life-Cycle.

Kinome from apicomplexan parasites is composed of eukaryotic protein kinases and Apicomplexa specific kinases, such as rhoptry kinases (ROPK). Ropk is a gene family that is known to play important roles in host–pathogen interaction in Toxoplasma gondii but is still poorly described in Eimeria tenella, the parasite responsible for avian coccidiosis worldwide. In the E. tenella genome, 28 ropk genes are predicted and could be classified as active (n = 7), inactive (incomplete catalytic triad, n = 12), and non-canonical kinases (active kinase with a modified catalytic triad, n = 9). We characterized the ropk gene expression patterns by real-time quantitative RT-PCR, normalized by parasite housekeeping genes, during the E. tenella life-cycle. Analyzed stages were: non-sporulated oocysts, sporulated oocysts, extracellular and intracellular sporozoites, immature and mature schizonts I, first- and second-generation merozoites, and gametes. Transcription of all those predicted ropk was confirmed. The mean intensity of transcription was higher in extracellular stages and 7–9 ropk were specifically transcribed in merozoites in comparison with sporozoites. Transcriptional profiles of intracellular stages were closely related to each other, suggesting a probable common role of ROPKs in hijacking signaling pathways and immune responses in infected cells. These results provide a solid basis for future functional analysis of ROPK from E. tenella.

A bivalent vaccine against avian necrotic enteritis and coccidiosis.

In this study, we attempted to design a recombinant vaccine harbouring domain with a key role in enterocyte attachment and cell invasion in necrotic enteritis (NE) and coccidiosis. In this study, we investigated whether a recombinant protein consisting of necrotic enteritis B-like toxin, C-terminal domain of alpha-toxin, apical membrane antigen 1 (AMA1), and Rhoptry neck protein 2 (RON2) which we call "NeCoVac" hereafter, can improve protection against both diseases compared to vaccination with each antigen in previous studies. Birds intestinal lesion scores and specific antibody levels were measured to determine protection after oral gavage challenges with virulent Clostridium perfringens and LIVACOX® T. Birds immunized with NeCoVac were protected up to 84% against NE and coccidiosis compared to unimmunized and even positive groups (groups treated with LIVACOX® T [coccidiosis live vaccine] and tylosin as routine veterinary interventions) (p<0.05). Our findings suggest that vaccination with NeCoVac is highly efficient in protecting birds from NE, coccidiosis and a combination of both diseases. The present study is the first one to describe the combinatorial use of AMA1 and RON2 against coccidiosis, and the first report using NeCoVac against NE and coccidiosis together.

In Vitro Assessment of Anticoccidials: Methods and Molecules

Simple SummaryCoccidiosis is a major problem in poultry production, leading to significant economic losses. Due to the outbreak of resistance to the available treatments, research is focusing on finding new molecules that work against the pathogen. Botanical compounds represent promising alternatives, but reliable in vitro tests are needed for their screening and to understand their mechanism of action. Research in vitro involves studies on the environmental phase of the parasite and studies on the endogenous development, which occurs inside the host cells and that requires cell cultures or in ovo models to be studied. This review aims to summarize the protocols that have been successfully applied so far, as well as to suggest potential cues to improve research on this field. Moreover, as the surge of botanicals as anticoccidial molecules is on the rise, the intent is to provide an overview of the methods to assess their effectiveness in vitro in comparison with conventional drugs.Avian coccidiosis is a disease causing considerable economic losses in the poultry industry. It is caused by Eimeria spp., protozoan parasites characterized by an exogenous–endogenous lifecycle. In vitro research on these pathogens is very complicated and lacks standardization. This review provides a description of the main in vitro protocols so far assessed focusing on the exogenous phase, with oocyst viability and sporulation assays, and on the endogenous phase, with invasion and developmental assays in cell cultures and in ovo. An overview of these in vitro applications to screen both old and new remedies and to understand the relative mode of action is also discussed.

Full-length transcriptome sequence analysis of Eimeria necatrix unsporulated oocysts and sporozoites identifies genes involved in cellular invasion

Eimeria necatrix is one of the most pathogenic chicken coccidia and causes avian coccidiosis, an enteric disease of major economic importance worldwide. Eimeria parasites have complex developmental life cycles, with an exogenous phase in the environment and an endogenous phase in the chicken intestine. Oocysts excreted by chickens rapidly undergo meiosis and cell division to form eight haploid sporozoites (SZ). SZ liberated from sporocysts in the chicken intestine migrate to their preferred site of development to initiate cellular invasion. To date, almost nothing is known about the proteins that mediate parasite invasion in E. necatrix. In order to discover genes with functions involved in cellular invasion, the transcriptome profiles of E. necatrix unsporulated oocysts (UO) and SZ were analyzed using a combination of third-generation single-molecule real-time sequencing (TGS) and second-generation sequencing (SGS) followed by qRT-PCR validation. Correction of TGS long reads by SGS short reads resulted in 34,932 (UO) and 23,040 (SZ) consensus isoforms. After subsequent assembly, a total of 4949 and 4254 genes were identified from UO and SZ libraries, respectively. A total of 8376 genes were identified as differentially expressed genes (DEGs) between SZ and UO. Compared to UO, 4057 genes were upregulated and 4319 genes were downregulated in SZ. Approximately 1399 and 1758 genes were defined as stage-specific genes in SZ and UO, respectively. Gene Ontology (GO) classification and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that 2978 upregulated SZ genes were clustered into 29 GO terms, and 857 upregulated SZ genes were associated with 26 KEGG pathways. We also predicted a further 50 upregulated SZ genes and 73 upregulated UO genes encoding microneme proteins, apical membrane antigens, rhoptry neck proteins, rhoptry proteins, dense granule proteins, heat shock proteins, calcium-dependent protein kinases, cyclin-dependent kinases, cGMP-dependent protein kinase, and glycosylphosphatidylinositol-anchored surface antigens. Our data reveal new features of the E. necatrix transcriptional landscape and provide resources for the development of novel vaccine candidates against E. necatrix infection.

Brief History and Advancements of Vaccination Against Avian Coccidiosis: A Review

Coccidiosis is a major protozoal disease that severely affects livestock and other animals, especially poultry. Seven species of Eimeria cause avian coccidiosis in poultry and evolve from the epithelial cells of intestine, readily induce illness and cause death to a varying extent. Prophylactic chemotherapy was a dominant choice for the control of coccidiosis but resistance to the drug was a major factor of therapy failure. Protective immunity was produced in chickens with any of Eimeria species as only species-specific immunity can be produced by recently used vaccines. Attenuation can be achieved by the serial passages in all seven Eimeria species. In chicken, the first attempt against coccidiosis caused the introduction of live oocysts, the basis of which led to the discovery of first live attenuated commercial vaccine, Paracox1. As the emerged recombinant vaccines were replaced as a first choice, there is still a dire need to do more work on new techniques like DNA vaccine formulation along with the role of dendritic cells to produce immunity and cross-protection against avian coccidiosis. This article describes step-by-step developments in the vaccination process from the last 70 years along with a brief discussion on novel techniques to induce immunity against coccidiosis.

Anticoccidial effects of Phyllanthus emblica (Indian gooseberry) extracts: Potential for controlling avian coccidiosis.

The protozoan parasite Eimeria causes avian coccidiosis, impacting the poultry industry worldwide. Resistance development to current anticoccidials are a concern and cost effective, environmentally friendly alternatives are needed. Anti-malarial effects of Phyllanthus emblica encouraged us to investigate its anticoccidial effects. Aqueous extracts and dried-powder of P. emblica leaf and fruit were tested for effect(s) on oocyst sporulation in vitro and oocyst infectivity in vivo. Eimeria tenella oocysts were randomly assigned to groups and treated with different concentrations (0.001, 0.1, 1, 5, 25, 50 and 100%) of P. emblica crude extracts in triplicates for three repeats. Sporulated, unsporulated, deformed and lysed oocysts were recorded at 24, 48 and 72h. Broiler chicks (21days old) were randomly assigned into four groups with 5 chicks each and experimentally infected on Day 0 with 1×104 oocysts/bird: (A) infected and un-supplemented diet, (B) infected and supplemented diet (P. emblica powder 1g/bird/day), (C) infected with P. emblica-treated oocysts and un-supplemented diet and (D) infected and diet supplemented only from day14. In vivo experiments were terminated on day28. Significant sporulation inhibition and oocyst lysis (p<0.05) in vitro were observed in a concentration-dependent with P. emblica treatment. In in vivo experiments, group B showed the highest weight gain, lowest fecal oocyst excretion and mildest histopathological lesions. Extracts of P. emblica remarkably inhibited oocyst sporulation, reduced the oocyst infectivity and lowered the fecal oocyst excretion, and reduced the pathogenicity of E. tenella in chickens. Therefore, P. emblica extract demonstrates great potential to be an effective alternative anticoccidial agent.

Protective Efficacy of Rhomboid-Like Protein 3 as a Candidate Antigen Against Eimeria maxima in Chickens.

Avian coccidiosis brings tremendous economic loss to the poultry industry worldwide. The third generation vaccine, including subunit and DNA vaccines, exhibited promising developmental prospects. In a previous study, we found rhomboid-like protein 3 of Eimeria maxima (EmROM3) was involved in infections by Eimeria species. However, the protective efficacy of EmROM3 against Eimeria maxima (E. maxima) remains unknown. In this study, chickens were intramuscularly immunized with the recombinant protein EmROM3 (rEmROM3) or pVAX1-EmROM3 to determine the EmROM3-induced immune response. The induced humoral immune response was determined by measuring serum IgG antibody levels in immunized chickens. The induced cellular immune response was detected by measuring the transcription level of immune related cytokines and the proportion of T cell subsets of the immunized chickens. Finally, the protective efficacy of the EmROM3 vaccine against E. maxima was evaluated by immunization-challenge trials. Results revealed that the purified rEmROM3 reacted with chicken anti-E. maxima serum. The recombinant plasmid of pVAX1-EmROM3 was transcribed and translated in the injected muscle from the vaccinated chickens. In experimental groups, the IgG titers, proportions of CD4+ and CD8+ T cells, and transcription level of splenic cytokines were significantly increased compared with the control groups. The immunization-challenge trial revealed that immunization with rEmROM3 or pVAX1-EmROM3 led to restored weight gain, alleviated enteric lesion, decreased oocyst output as well as the higher anticoccidial index (ACI), indicating partial protection against E. maxima. These results indicate that EmROM3 is an effective candidate antigen for developing novel vaccines against infection by E. maxima.

Survey of coccidia on commercial broiler farms in Colombia: frequency of Eimeria species, anticoccidial sensitivity, and histopathology

Avian coccidiosis continues to be one of the costliest diseases of commercial poultry. Understanding the epidemiology of Eimeria species in poultry flocks and the resistance profile to common anticoccidials is important to design effective disease prevention and control strategies. This study examined litter samples to estimate the prevalence and distribution of Eimeria species among broiler farms in 4 geographic regions of Colombia. A total of 245 litter samples were collected from 194 broiler farms across representative regions of poultry production between March and August 2019. The litter samples were processed for oocysts enumeration and speciation after sporulation. End-point polymerase chain reaction (PCR) analysis was conducted to confirm the presence of Eimeria species. Anticoccidial sensitivity was determined with 160 Ross AP males in 5 treatment groups: noninfected, nonmedicated control (NNC), infected, nonmedicated control (INC), infected salinomycin treated (SAL, dose: 66 ppm), infected diclazuril treated (DIC, dose: 1 ppm), and infected methylbenzocuate-Clopidol treated (MET.CLO, dose: 100 ppm), All birds were orally inoculated with 1 × 106 sporulated oocysts using a 1 mL syringe, except for the NNC- group who received 1ml of water.Eimeria spp. were found in 236 (96.3%) out of 245 individual houses, representing 180 (92.8%) out of 194 farms. Eimeria acervulina was the most prevalent species (35.0%) followed by Eimeria tenella (30.9%), Eimeria maxima (20.4%), and other Eimeria spp. (13.6%). However, mixed species infections were common, with the most prevalent combination being mixtures of E. acervulina, E. maxima, E. tenella, and other species in 31.4% of the Eimeria-positive samples. PCR analysis identified E. acervulina, E. maxima, E. tenella, Eimeria necatrix, Eimeria mitis, and Eimeria praecox with variable prevalence across farms and regions. Anticoccidial sensitivity testing of strains of Eimeria isolated from 1 region, no treatment difference (P > 0.05) was observed in final weight (BW), weight gain (BWG) or feed conversion (FCR). For the global resistance index (GI) classified SAL and MET.CLO as good efficacy (85.79 and 85.49, respectively) and DIC as limited efficacy (74.52%). These results demonstrate the ubiquitous nature of Eimeria spp. and identifies the current state of sensitivity to commonly used anticoccidials in a region of poultry importance for Colombia.

Comparative evaluation of single or combined anticoccidials on performance, antioxidant status, immune response, and intestinal architecture of broiler chickens challenged with mixed Eimeria species

Poultry production faces several threats and challenges, one of the most important of which is avian coccidiosis which causes annual losses exceeding US$ 3 billion. Discovering new drugs or combinations of existing anticoccidials has become inevitable to overcome the emergence of coccidiosis resistance. This study evaluated a new combination of maduramicin and diclazuril in comparison to the well-known product Maxiban72 which consisted of narasin and nicarbazin, and the single effect of monensin as treatments for avian coccidiosis. A total of 750 1-day-old Indian River broiler chicks were allocated equally into 5 experimental groups with 6 replicates each as follows: 1) negative unchallenged control group (NC) fed the basal diet; 2) positive control group (PC) received the basal diet and inoculated with Eimeria; 3) PC + 100 mg monensin sodium (Atomonsin)/kg diet (MS); 4) PC + 5 mg maduramicin ammonium (Madramycin) + 2.5 mg diclazuril (Atozuril)/kg diet (MMD); and 5) PC + 40 mg narasin + 40 mg nicarbazin (MaxibanT72)/kg diet (NN). Anticoccidials improved (P < 0.01) growth performance, dressing (%) and carcass yield of inoculated birds compared to untreated-inoculated ones. Erythrogram and leukogram parameters were affected by Eimeria challenge. Total protein, globulin, cholesterol, triglycerides, superoxide dismutase and glutathione peroxidase levels in PC birds' serum were reduced (P < 0.05) while their values of liver enzymes, malondialdehyde and catalase were elevated (P < 0.01) when compared to NC ones. Serum immunoglobulin A, and jejunal gene expressions of interleukin-6 and interferon gamma were increased (P < 0.05) in PC group compared to NC group. Anticoccidial drugs restored values of the aforementioned biomarkers near to those of NC. Jejunal architecture in inoculated birds was improved by the anticoccidial treatments in MS, MMD, and NN. Fecal oocyst counts were significantly reduced in MMD, NN, and MS groups compared to PC group. Conclusively, although all examined anticoccidial drugs were effective in treating Eimeriosis, the anticoccidial combinations in MMD and NN groups were more effective than the single administration of MS in treating avian coccidiosis.

Anticoccidial effect of Origanum majoranum aqueous extract on Eimeria tenella-infected chicken.

Avian coccidiosis, an important protozoal disease of chicken triggered by coccidian protozoa of genus Eimeria, causes considerable economic losses to broiler producers. The study was designed to assess the efficiency of Origanum majoranum aqueous extract (OMAE) on E. tenella-infected broiler chicken. Birds were divided into four groups including: positive control (PC, challenged with 5×104 sporulated oocysts of E. tenella at the 12th day of age), PC+OMAE (challenged with E. tenella oocysts at the 12th day of age and received OMAE (125 mg/kg BW) orally, started at the 7th day of age, and continued for 14 consecutive days), OMAE (received OMAE (125 mg/kg BW) orally, at the 7th day of age, for 14 consecutive days), and negative control (received basal diet only). Anticoccidial efficacy of OMAE was evaluated by complete blood picture, serum chemistry, serum protein electrophoresis, antioxidants markers, cecal oocysts count, and cecal lesions score. Briefly, collected data indicated that supplementation of OMAE could increase antioxidants concentrations and improve changes in hematobiochemical parameters and serum protein fractions, as well as decrease cecal oocysts count and reduce cecal lesion scores in E. tenella-infected birds. In conclusion, OMAE restores oxidant-antioxidant balance, and its supplementation in broiler chicken can alleviate E. tenella-infection and reduce its severity.

Poly (D, L-lactide-co-glycolide) delivery system improve the protective efficacy of recombinant antigen TA4 against Eimeria tenella infection

Eimeria tenella is a protozoan parasite endemic in chickens and is one of the causative agents of avian coccidiosis. The aim of this research was to determine if poly (D, L-lactide-co-glycolide) (PLGA) nanoparticles carrying recombinant TA4 protein of E. tenella (rEtTA4) could improve the level of protective immunity against E. tenella challenge. Recombinant TA4 protein was expressed and purified. Poly (D, L-lactide-co-glycolide) loaded with rEtTA4 (PLGA-rEtTA4) nanoparticles was prepared and was delivered to 2-week-old layer chickens via intramuscular inoculation. Chickens injected with PBS and PLGA nanoparticles were served as control groups. The rEtTA4 and PLGA-rEtTA4 nanoparticles induced changes of serum cytokines, IgY levels, and T lymphocytes subpopulation, and the protective efficacy against E. tenella challenge was evaluated. Results showed that both rEtTA4 and PLGA-rEtTA4 vaccination groups induced significantly higher levels of specific EtTA4 IgY antibody and IL-17 and higher proportion of CD8+ T lymphocytes. However, no significant differences were observed in the proportion of CD4+ T lymphocytes compared with the PBS control. Chickens immunized with rEtTA4 and PLGA-rEtTA4 prominently increased the BW gains and decreased oocyst output compared with chickens immunized with PBS and PLGA after oral challenge with E. tenella. Poly (D, L-lactide-co-glycolide) encapsulated rEtTA4 nanoparticles–immunized chickens significantly induced higher levels of interferon gamma, IL-6, and IL-17 and a little bit higher proportion of CD8+ T lymphocytes compared with rEtTA4 subunit vaccine–immunized chickens. Thus, PLGA encapsulated rEtTA4 nanoparticles appeared to have great potential to enhance the immune response and improved the protective efficacy against E. tenella infection. Our results provided available protective subunit vaccine rEtTA4 and PLGA loaded with rEtTA4 nanoparticles against coccidiosis and suggested that PLGA nanoparticles could be an effective adjuvant to enhance the protective efficacy of rEtTA4 subunit vaccine.