Identification of Eimeria acervulina (Apicomplexa: Eimeriidae) infecting the broiler chicken Gallus gallus domesticus through morphology and molecular analyses.

Coccidiosis is an intestinal protozoan disease that affects the poultry industry worldwide. The severity of this disease varies depending on the identity of the infectious agents. Therefore, this study was carried out to identify the Eimeria species that affect broiler chickens, Gallus gallus domesticus, through morphological and molecular phylogenetic analyses. Twenty-five faecal samples were collected from the broiler chickens in a commercial poultry farm in Riyadh (Saudi Arabia). Using the floatation technique, faeces were examined microscopically for the Eimeria occurrence. Identification of Eimeria species was performed based on morphological criteria and molecular tools (DNA amplification for the partial small subunit ribosomal RNA (18S rRNA), internal transcribed spacer (ITS)-1, and mitochondrial cytochrome c oxidase I (COI) genes. In this study, 32% (8 out of 25) of collected samples were found to be positive for coccidiosis. After sporulation in potassium dichromate (K2 Cr2 O7 ), the sporulated oocysts were observed as ovoid and measured 18.37-23.19 µm (19.87) long and 15.07-18.67 µm (16.46) wide, with the anterior location of a polar granule and absence of micropyle. These Eimeria oocysts were assumed to size and shape characteristics of Eimeria acervulina. Molecular analysis was conducted on the sequences of the polymerase chain reaction products from the three genes studied (18S rRNA, ITS-1, and COI). At the three genes, results showed that the resultant sequences clustered with E. acervulina from different regions confirming morphological description. This study highlighted the importance of molecular techniques to detect avian Eimeria species more than the traditional morphology-based tool to optimise the appropriate anticoccidial strategies for long-term control in the studied area.

Protective Efficacy Induced by the Common Eimeria Antigen Elongation Factor 2 against Challenge with Three Eimeria Species in Chickens.

Avian coccidiosis arises from co-infection involving multiple Eimeria species, which could give rise to substantial economic losses in the global poultry industry. As a result, multivalent anticoccidial vaccines containing common Eimeria antigens offer considerable promise for controlling co-infection in clinical practice. In our previous study, Elongation factor 2 (EF2) was deemed as an immunogenic common antigen across various Eimeria species. This current investigation aimed to further assess the immunogenicity and protective efficacy of EF2 in recombinant subunit vaccine format against three Eimeria species. The EF2 gene cloned from Eimeria maxima (E. maxima) cDNA was designated as EF2 of E. maxima (EmEF2). The immunogenicity of the recombinant protein EmEF2 (rEmEF2) was assessed through Western blot analysis. The evaluation of the vaccine-induced immune response encompassed the determination of T lymphocyte subset proportions, cytokine mRNA transcription levels, and specific IgY concentrations in rEmEF2-vaccinated chickens using flow cytometry, quantitative real-time PCR (qPCR), and indirect enzyme-linked immunosorbent assay (ELISA). Subsequently, the protective efficacy of rEmEF2 was evaluated through vaccination and challenge experiments. The findings demonstrated that rEmEF2 was effectively recognized by the His-tag monoclonal antibody and E. maxima chicken antiserum. Vaccination with rEmEF2 increased the proportions of CD4+ and CD8+ T lymphocytes, elevated IL-4 and IFN-γ mRNA transcription levels, and enhanced IgY antibody levels compared to the control groups. Moreover, compared to the control groups, vaccination with rEmEF2 led to decreased weight loss, reduced oocyst outputs, and alleviated enteric lesions. Furthermore, in the rEmEF2-immunized groups, challenges with E. maxima and E. acervulina resulted in anticoccidial index (ACI) scores of 166.35 and 185.08, showing moderate-to-excellent protective efficacy. Nevertheless, challenges with E. tenella and mixed Eimeria resulted in ACI scores of 144.01 and 127.94, showing low protective efficacy. In conclusion, EmEF2, a common antigen across Eimeria species, demonstrated the capacity to induce a significant cellular and humoral immune response, as well as partial protection against E. maxima, E. acervulina, and E. tenella. These results highlight EmEF2 as a promising candidate antigen for the development of multivalent vaccines targeting mixed infections by Eimeria species.

Thyme, Oregano, and Garlic Essential Oils and Their Main Active Compounds Influence Eimeria tenella Intracellular Development.

Coccidiosis poses a significant challenge in poultry production and is typically managed with ionophores and chemical anticoccidials. However, the emergence of drug resistance and limitations on their use have encouraged the exploration of alternative solutions, including botanical compounds and improvements in in vitro screening methods. Prior research focused only on the impact of these alternatives on Eimeria invasion, with intracellular development in cell cultures receiving limited attention. This study assessed the impact of thyme (Thymus vulgaris), oregano (Origanum vulgare), and garlic (Allium sativum) essential oils, as well as their bioactive compounds, on the initial phase of schizogony in Madin-Darby bovine kidney cells, comparing their effectiveness to two commercially used anticoccidial drugs. Using image analysis and quantitative PCR, the study confirmed the efficacy of commercial anticoccidials in reducing invasion and schizont formation, and it found that essential oils were equally effective. Notably, thymol and carvacrol exhibited mild inhibition of intracellular replication of the parasite but significantly reduced schizont numbers, implying a potential reduction in pathogenicity. In conclusion, this research highlights the promise of essential oils and their bioactive components as viable alternatives to traditional anticoccidial drugs for mitigating coccidiosis in poultry, particularly by disrupting the intracellular development of the parasites.

Transcriptome analysis of differentially expressed circRNAs miRNAs and mRNAs during the challenge of coccidiosis.

Avian coccidiosis is a common enzootic disease caused by infection of Eimeria species parasites. It causes huge economic losses in the global poultry industry. Current control using anticoccidial drugs or vaccination is limited due to drug resistance and the relatively high cost of vaccines. Improving host genetic resistance to Eimeria species is considered an effective strategy for improved control of coccidiosis. Circular RNAs (circRNAs) have been found to function as biomarkers or diagnoses of various kinds of diseases. The molecular biological functions of circRNAs, miRNAs, and mRNAs related to Sasso chicken have not yet been described during Eimeria species challenge. In this study, RNA-seq was used to profile the expression pattern of circRNAs, miRNAs, and mRNAs in spleens from Eimeria tenella-infected and non-infected commercial dual-purpose Sasso T445 breed chickens. Results showed a total of 40 differentially expressed circRNAs (DEcircRNAs), 31 differentially expressed miRNAs (DEmiRNAs), and 820 differentially expressed genes (DEmRNAs) between infected and non-infected chickens. Regulatory networks were constructed between differentially expressed circRNAs, miRNAs, and mRNAs to offer insights into the interaction mechanisms between chickens and Eimeria spp. Functional validation of a significantly differentially expressed circRNA, circMGAT5, revealed that circMGAT5 could sponge miR-132c-5p to promote the expression of the miR-132c-5p target gene monocyte to macrophage differentiation-associated (MMD) during the infection of E. tenella sporozoites or LPS stimulation. Pathologically, knockdown of circMGAT5 significantly upregulated the expression of macrophage surface markers and the macrophage activation marker, F4/80 and MHC-II, which indicated that circMGAT5 might inhibit the activation of macrophage. miR-132c-5p markedly facilitated the expression of F4/80 and MHC-II while circMGAT5 could attenuate the increase of F4/80 and MHC-II induced by miR-132c-5p, indicating that circMGAT5 exhibited function through the circMGAT5-miR-132c-5p-MMD axis. Together, our results indicate that circRNAs exhibit their resistance or susceptive roles during E. tenella infection. Among these, circMGAT5 may inhibit the activation of macrophages through the circMGAT5-miR-132c-5p-MMD axis to participate in the immune response induced by Eimeria infection.

Comparison of endogenous development, invasion ability and apoptotic features between diclazuril resistant and sensitive strains of Eimeria tenella.

Diclazuril (DIC) is widely used in the poultry industry to control coccidiosis. However, drug resistance makes it less effective, and the underlying mechanism remains unclear. One DIC-resistant E. tenella (RE) isolate and one sensitive E. tenella (SE) isolate were used to compare the differences in their endogenous development, pathogenicity, invasion-related gene expression and apoptotic characteristics. Chickens were allocated into four groups to receive RE or SE strain and their corresponding DIC treatment or not. Caeca tissues were sampled at 96 h, 120 h and 144 h post-infection (PI) for pathological analysis. Meanwhile, second-generation merozoites (Mz2) were separated at 120 h PI to detect alterations in mitochondrial membrane potential (MMP), apoptotic rate and caspase-3 activity and mRNA expression of protein phosphatase 5 (PP5), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), actin depolymerizing factor (ADF) and microneme proteins (MICs). Haematoxylin and eosin staining revealed that DIC treatment strictly blocked the development of the SE strain but slightly affected the RE strain. Meanwhile, the number of SE Mz2 and their MMP decreased at the same time the apoptotic rate increased after DIC treatment. Real-time quantitative PCR and caspase-3 activity studies demonstrated that Mz2 from the RE strain had higher mRNA expression of ADF and MICs along with no significant changes in GAPDH and caspase-3 activity under DIC pressure compared to its control; in contrast, the mRNA expression of ADF, MICs and PP5 was markedly suppressed in Mz2 from SE with upregulated caspase-3 activity and GAPDH transcription. In addition, the mRNA expression of GAPDH and PP5 in Mz2 from RE was remarkably higher than that of SE. Taken together, the higher mRNA expression of invasion-related genes and almost unaffected endogenous development provide a better understanding of coccidian resistance to DIC.

Toltrazuril-Loaded Polymeric Nanocapsules as a Promising Approach for the Preventive Control of Coccidiosis in Poultry.

Coccidiosis is a disease caused by intracellular protozoan parasites of the genus Eimeria that affect the intestinal tract of poultry. However, strain resistance and drug residue in the carcass have drawn the attention of the productive sector. The nanotechnology can improve the biological effect of drugs, reducing of administered doses and toxic effects. Due to this, toltrazuril-load polymeric nanoparticles based on Eudragit® S100 (NCt) or poly-ε-caprolactone (LNCt) were developed to prevent coccidiosis in broilers. Nanoformulations were produced and showed homogeneous particle diameter distribution in the nanometer range (z-average and D (4.3) < 200 nm), negative zeta potential (<-8.93 mV), drug content ~100%, and encapsulation efficiency >90%. Cell viability assays using avian fibroblasts showed that LNCt presented no relevant toxicity up to 72 h. LNCt was then prophylactically administrated to chicken followed by challenge with Eimeria oocysts. The evaluation of the small intestine and cecum showed that the treatment with LNCt (3.5 mg/kg/day) in drinking water reduced the lesion scores and oocysts excretion, similar to the reference medicine containing toltrazuril (Baycox®, 7 mg/kg/day). The current study shows the potential protective use of nanoencapsulating anticoccidial drugs as a promising approach for the control of coccidiosis in poultry.

In vitro inhibitory activities of sugarcane extract on avian Eimeria sporozoites.

The current in vitro study aimed to investigate the effects of a processed sugarcane extract on the viability of avian Eimeria sporozoites. Treatments were applied to hatched sporozoites: 1) without additives (no-treatment control); 2) with ethanol; 3) with salinomycin; 4) with Polygain™. All treatments were incubated in RPMI media containing live sporozoites at 37 °C for 14 h and then the number of viable sporozoites were counted. Compared to the no-treatment control, Polygain™ decreased (P < 0.001) the counts of E. maxima, E. acervulina, E. bruneti, and E. mitis sporozoites to a level similar to salinomycin (P > 0.05). In conclusion, Polygain™ could be a potential candidate as an anticoccidial agent.

Molecular characterization of 60S ribosomal protein L12 of E. tenella.

This study analyzed the large-subunit (60S) ribosomal protein L12 of Eimeria tenella (Et60s-RPL12). A full-length cDNA was cloned, and the recombinant protein was expressed in E. coli BL21 and inoculated in rabbits to produce the polyclonal antibody. Quantitative real-time polymerase chain reaction and western blotting were used to analyze the transcription levels of Et60s-RPL12 and translation levels in different developmental stages of E. tenella. The results showed that the mRNA transcription level of Et60s-RPL12 was highest in second-generation merozoites, whereas the translation level was highest in unsporulated oocysts. Indirect immunofluorescence showed that Et60s-RPL12 was localized to the anterior region and surface of sporozoites, except for the two refractile bodies. As the invasion of DF-1 cells progressed, fluorescence intensity was increased, and Et60s-RPL12 was localized to the parasitophorous vacuole membrane (PVM). The secretion assay results using staurosporine indicated that this protein was secreted, but not from micronemes. The role of Et60s-RPL12 in invasion was evaluated in vitro. The results of the invasion assay showed that polyclonal antibody inhibited host cell invasion by the parasite, which reached about 12%. However, the rate of invasion was not correlated with the concentration of IgG.

Anticoccidial effect of Fructus Meliae toosendan extract against Eimeria tenella.

CONTEXT: Fructus Meliae toosendan extracts (FMTE) have a good therapeutic effect on coccidiosis, but there is no relevant research on its prophylactic effect on coccidiosis. OBJECTIVE: This study comprehensively evaluates the anticoccidial effect of FMTE. MATERIALS AND METHODS: In vitro, the unsporulated oocysts were treated with serial dilutions of FMTE and incubated for 7 d, and the sporulated oocysts were counted for calculating the median lethal concentration (LC50) of FMTE. In vivo, 180 10-day-old broiler chickens free of coccidiosis were weighted and randomly distributed into six groups: normal group, untreated group, 4 protective groups (positive group and three FMTE groups). From day 10 to day 21, chickens in the three FMTE groups were pre-treated with FMTE at the dosage of 2.5, 5 and 10 g/kg/d, respectively, and chickens in the positive group were pre-treated with qiuliling (10 g/kg/d). On day 14, chickens in all groups except the normal group were orally infected with 1.5 × 104 sporulated oocysts. The clinical symptoms were observed from day 10 to day 21, the anticoccidial index (ACI), tissue lesions, and intestinal microflora were determined on day 21. RESULTS: FMTE showed anti-sporulation effect against E. tenella and the LC50 value was 245.83 µg/mL in vitro. In vivo, FMTE at the dosage of 10 g/kg/d was effective against E. tenella infection, and its ACI value was 162.56, which was higher than the value of positive drug qiuliling (128.81). Discussion and conclusions: FMTE have potent anticoccidial effects, and it presents an alternative anticoccidial agent for avian coccidiosis control.

Pleurotus ostreatus extract inhibits Eimeria species development in naturally infected broiler chickens.

Coccidian organisms of poultry have proved very hard to control due to their abilities to resist most anticoccidial drugs. Pleurotus ostreatus (Fr.) Jacq. ex (Pleurotaceae), a medicinal mushroom, was investigated in vivo against Eimeria spp. Ninety-six broilers (day-old) naturally infected with Eimeria spp. were divided into eight groups (12 birds per group). Group A was infected untreated (negative control) and group B was treated with toltrazuril (positive control) while groups C-H were gavaged with graded doses of P. ostreatus extract at 100, 200, 300, 400, 500, and 600 mg/kg, respectively. The phytochemical analysis of the aqueous extract of P. ostreatus which revealed saponins, flavonoids, anthraquinones, and alkaloids was evaluated for anticoccidial activity by assessing the inhibition of oocyst output, lesion score, faecal score, weight differences, haematological parameters, and leucocyte differential counts. The acute toxicity study showed extract of P. ostreatus to be non-toxic at 600 mg/kg. The weight of the groups treated with the extracts and toltrazuril increased significantly (P < 0.05) compared with the untreated control. Treated groups significantly (P < 0.05) reduced oocyst output except groups C and D. The therapeutic best-fit ED50 value for the extract was 448 mg/kg. The post-treatment mean packed cell volume and red blood cell count were significantly higher (P < 0.05) than the untreated group, while the WBC count was significantly higher (P < 0.05) in the untreated group. Pleurotus ostreatus therefore could be a potential source of new anticoccidial medicine which could find application in the control of avian coccidiosis.

Incorporation of a recombinant Eimeria maxima IMP1 antigen into nanoparticles confers protective immunity against E. Maxima challenge infection.

The purpose of this study was to determine if conjugating a recombinant Eimeria maxima protein, namely EmaxIMP1, into 20 nm polystyrene nanoparticles (NP) could improve the level of protective immunity against E. maxima challenge infection. Recombinant EmaxIMP1 was expressed in Escherichia coli as a poly-His fusion protein, purified by NiNTA chromatography, and conjugated to 20 nm polystyrene NP (NP-EmaxIMP1). NP-EMaxIMP1 or control non-recombinant (NP-NR) protein were delivered per os to newly-hatched broiler chicks with subsequent booster immunizations at 3 and 21 days of age. In battery cage studies (n = 4), chickens immunized with NP-EMaxIMP1 displayed complete protection as measured by weight gain (WG) against E. maxima challenge compared to chickens immunized with NP-NR. WG in the NP-EMaxIMP1-immunized groups was identical to WG in chickens that were not infected with E. maxima infected chickens. In floor pen studies (n = 2), chickens immunized with NP-EMaxIMP1 displayed partial protection as measured by WG against E. maxima challenge compared to chickens immunized with NP-NR. In order to understand the basis for immune stimulation, newly-hatched chicks were inoculated per os with NP-EMaxIMP1 or NP-NR protein, and the small intestine, bursa, and spleen, were examined for NP localization at 1 h and 6 h post-inoculation. Within 1 h, both NP-EMaxIMP1 and NP-NR were observed in all 3 tissues. An increase was observed in the level of NP-EmaxIMP1 and NP-NR in all tissues at 6 h post-inoculation. These data indicate that 20 nm NP-EmaxIMP1 or NP-NR reached deeper tissues within hours of oral inoculation and elicited complete to partial immunity against E. maxima challenge infection.

Quantitative real-time PCR (qPCR) for Eimeria tenella replication--Implications for experimental refinement and animal welfare.

The Eimeria species are highly pathogenic parasites of chickens. Research aimed at reducing their impact is hindered by a lack of non-subjective, quantitative, tools to measure parasite replication in the host. The time-consuming, and often time-sensitive, nature of existing approaches precludes their use in large-scale genetic, epidemiological, and evolutionary analyses. We have used quantitative real-time PCR (qPCR) to accurately quantify Eimeria tenella in chicken tissue and shown this to be more efficient and sensitive than traditional methodologies. We tested four chicken-specific reference qPCR assays and found beta-actin (actb) to be optimal for sample normalisation. In an experimental setting, chickens were inoculated with 500, 1500, or 4500 E. tenella oocysts and parasite replication and the impact of infection measured by i) qPCR analysis of DNA extracted from caecal tissues collected at five and eight days post-infection (dpi), ii) faecal oocyst counts (FOCs) on samples taken from six to eight dpi, and iii) lesion scoring on caeca collected post-mortem at five and eight dpi. Quantitative real-time PCR test results indicated a significant dose-dependent increase in parasite numbers among study groups for samples collected five dpi (i.e., prior to gametogony) (R(2)=0.994) (p<0.002) but not in those from day eight (after most oocyst shedding) (R(2)=0.006) (p>0.379). A strong dose-dependent increase in parasite replication and severity of infection was also revealed by FOC (R(2)=0.997) and lesion scoring. Importantly, qPCR offers substantial improvements for animal welfare via improved statistical power and reduced group sizes in experimental studies. The described qPCR method overcomes subjective limitations of coproscopic quantification, allows reproducible medium- to high-throughput examination of tissues, faeces, and oocysts, and is a valuable tool for determining the impact of Eimeria infections in both experimental and field settings.

Anticoccidial effects of a novel triazine nitromezuril in broiler chickens.

The anticoccidial efficacy of 2-(3-methyl-4-(4-nitrophenoxy)phenyl)-1,2,4-triazine-3,5(2H,4H)-dione (nitromezuril, NZL), a novel triazine compound, was evaluated in three different studies under experimental conditions. The anticoccidial efficacy was chiefly evaluated using the anticoccidial index (ACI). The resistance level was determined by calculating ACI, percentage optimum anticoccidial activity (POAA), reduction in lesion scores (RLS) and relative oocyst production (ROP). In the dose determination study (study A), NZL was added to the diet at doses of 1, 2, 3, 4, 5 and 6 mg/kg to test its efficacy against coccidiosis caused by Eimeria tenella. Groups treated with NZL 1mg/kg feed could observe the faecal dropping scores and caecal lesions. ACIs of NZL-treated groups reached 179-199. In the study on the anticoccidial efficacy of 3mg/kg NZL in the diet (study B), only a few faecal oocysts and slight lesions were observed. NZL significantly promoted weight gain (WG) and reduced lesion scores (LS) compared to controls receiving diclazuril (DZL) (P<0.05). ACIs of NZL-treated groups were 193, 192, 191 and 163 for E. tenella, Eimeria necatrix, Eimeria acervulina and Eimeria maxima, respectively, whereas those of DZL-treated groups were 185, 176, 176 and 148. In the cross-drug resistance study (study C), ACIs of NZL and toltrazuril (TZL)-treated groups ranged from 188 to 204, which were significantly higher than those of DZL-treated groups (P<0.05). NZL- and TZL-treated groups were sensitive to experimentally induced DZL-resistant E. tenella, whereas DZL-treated groups showed complete resistance. No cross-resistance was observed between DZL and NZL or TZL. Based on the abovementioned studies, it was concluded that diets containing 3 mg/kg NZL had an excellent efficacy in preventing coccidiosis in broiler chickens. The activity of 3mg/kg NZL in the diet was equal or superior to that of 1 mg/kg DZL. These results are of great significance for the future applications of NZL; however, its actual mechanism of action remains unknown. NZL is a potential novel anticoccidial agent suitable for further development.

Desinfecção com amônia quaternária associada à fermentação não potencializa o controle de coccidiose em cama de frango / Disinfection with quaternary ammonia associated with fermentation enhances do not control coccidiosis in poultry litter

O objetivo deste estudo foi avaliar se o uso da desinfecção com amônia quaternária associada à fermentação potencializa o controle de oocistos na cama de frangos de corte. A cama de frango reutilizada passou pela fermentação e/ou desinfecção com amônia quaternária no intervalo sanitário de 15 dias entre dois alojamentos sucessivos de 160 frangos cada. No 1° alojamento, as aves foram inoculadas, por via oral, com 3x10³ oocistos de Eimeria acervulina aos 12 dias de idade. Com a saída desse lote, a área de alojamento foi dividida em 4 boxes de 5m² cada. Cada box representou um grupo de tratamento da cama: grupo 1: a cama não passou por tratamento (controle); grupo 2: fermentação; grupo 3: desinfecção; e grupo 4: desinfecção associada à fermentação. No 2° alojamento, outras 160 aves foram divididas nos boxes. Foram realizadas contagens de oocistos por grama de fezes (OOPG), determinação do período pré patente, observação de lesões intestinais, avaliação de ganho de peso e da conversão alimentar. Os resultados demonstraram que não houve redução do número de oocistos recuperados da cama somente desinfetada (G3: 2,26x10(6)) em relação à cama não tratada (G1: 2,54x10(6)). Os grupos em que a cama foi fermentada (G2 e G4) apresentaram significativa redução do número de oocistos em relação ao grupo controle, no entanto, a cama fermentada e desinfetada (G4: 0,76x10(6)) não apresentou diferença significativa na redução do número de oocistos quando comparada à cama somente fermentada (G2: 0,67x10(6)). Conclui-se que a desinfecção com amônia quaternária associada à fermentação da cama não potencializa o controle da coccidiose aviária.

The objective of this study was to evaluate if the use of quaternary ammonium associated with the fermentation cause synergism in control of oocysts of poultry litter. The poultry litter recycled went through fermentation and/or disinfection with quaternary ammonium in sanitary interval between two successive housings of 160 chickens each. In the first housing, the birds were inoculated orally with 3x10³ Eimeria acervulina oocysts at 12 days old. With the removal of these birds, the housing area was divided into four pens of 5m² each. Each pen represents a treatment group of the litter: group 1: untreated (control), group 2: fermentation, group 3: disinfection and group 4: disinfection and fermentation. It was counted oocysts per gram of faeces (OOPG), determination of pre patent, intestinal lesions score, weight gain and feed conversion. The results showed no reduction in the number of oocysts recovered from the single litter only (G3: 2.26x10(6)) in relation to untread litter (G1: 2.54x10(6)). Groups in which the litter was fermented (G2 and G4) showed significant reduction of oocysts in the control group, however, the fermented and desinfected litter (G4: 0.76x10(6)) showed no significant difference in reducing the number of oocysts compared to single fermented litter (G2: 0.67x10(6)). It follows that the desinfection with quaternary ammonium associated to fermentacion of the litter does not potentiate the control of avian coccidiosis.

Sensitive and specific identification by polymerase chain reaction of Eimeria tenella and Eimeria maxima, important protozoan pathogens in laboratory avian facilities.

Eimeria tenella and Eimeria maxima are important pathogens causing intracellular protozoa infections in laboratory avian animals and are known to affect experimental results obtained from contaminated animals. This study aimed to find a fast, sensitive, and efficient protocol for the molecular identification of E. tenella and E. maxima in experimental samples using chickens as laboratory avian animals. DNA was extracted from fecal samples collected from chickens and polymerase chain reaction (PCR) analysis was employed to detect E. tenella and E. maxima from the extracted DNA. The target nucleic acid fragments were specifically amplified by PCR. Feces secreting E. tenella and E. maxima were detected by a positive PCR reaction. In this study, we were able to successfully detect E. tenella and E. maxima using the molecular diagnostic method of PCR. As such, we recommended PCR for monitoring E. tenella and E. maxima in laboratory avian facilities.