Abstract
Intestinal protozoan infections such as Eimeria spp. and Histomonas meleagridis remain major challenges to turkey production, leading to impaired feed efficiency, intestinal damage, and mortality. Accurate quantification of intestinal lesions is critical for evaluating disease severity and treatment efficacy; however, traditional lesion scoring systems are subjective and limited in detecting subtle or microscopic tissue changes. This research note introduces the use of Evans Blue Dye (EBD) as an objective, quantitative tool to assess intestinal and hepatic lesions in turkeys experimentally challenged with Eimeria or H. meleagridis. A total of one hundred eighty 1-day-old male poults were assigned to non-challenged (NC), Eimeria-challenged (EM), or H. meleagridis-challenged (HM) groups with five replicates per group and reared for 28 days. Evans Blue Dye (10 mg/kg BW) was injected intravenously one hour before necropsy, and dye concentrations were quantified spectrophotometrically in liver, duodenum, jejunum, and ceca tissues. HM poults showed the poorest growth performance and the highest liver and cecal lesion scores (P < 0.001), with EBD concentrations approximately 150% higher than those in controls, indicating severe vascular leakage and tissue damage caused by Histomonas challenge. EM poults exhibited moderate intestinal lesions and corresponding EBD increases in the duodenum and jejunum, consistent with species-specific invasion patterns. Overall, EBD concentration strongly reflected lesion severity and distribution, supporting its use as an objective and reproducible marker for evaluating protozoan-induced intestinal injury in poultry. This method minimizes observer bias and enhances sensitivity in detecting mild or microscopic lesions, offering a valuable measurement tool over conventional lesion scoring systems.
Keywords: Evans Blue Dye, Histomonas meleagridis, Eimeria, Lesion score
Introduction
Intestinal health plays a critical role in nutrient absorption, growth performance, and overall welfare of commercial turkeys. Among the enteric pathogens affecting turkey production, protozoan parasites such as Eimeria spp. and Histomonas meleagridis are of particular concern. Eimeria species cause coccidiosis, leading to impaired feed efficiency, intestinal damage, and predisposition to secondary infections. Similarly, H. meleagridis, the causative agent of histomoniasis (blackhead disease), induces severe necrotic lesions in the ceca and liver, resulting in high morbidity and mortality. Both pathogens compromise gut integrity and remain major challenges for sustainable turkey production worldwide (McDougald et al., 2020).
Quantifying intestinal damage is critical for evaluating disease progression and assessing the efficacy of prophylactic or therapeutic interventions against protozoan infections in poultry. Historically, lesion scoring has been the standard approach for both Eimeria and Histomonas meleagridis infections, offering qualitative or semi-quantitative assessments of tissue pathology. For coccidiosis, Johnson and Reid (1970) first introduced a systematic scoring method to quantify intestinal lesions in chickens, which was later adapted for turkeys. More recently, Gadde et al. (2020) described the pathology associated with the three major Eimeria species infecting turkeys (E. adenoeides, E. gallopavonis, and E. meleagrimitis), illustrated characteristic lesions with color photographs, and proposed a species-specific scoring system to standardize severity evaluation. Similarly, lesion scoring for H. meleagridis has been primarily based on gross visual inspection of cecal and hepatic lesions. McDougald and Hu (2001) reported a 0–4 scale system for histomoniasis, which although considered reasonably consistent when used by the same observer remains subjective and dependent on evaluator experience.
While these traditional scoring methods have provided valuable qualitative frameworks, they are limited by observer bias, inter-individual variability, and low sensitivity to subtle or subclinical tissue damage. Moreover, they do not account for microscopic mucosal disruption or vascular leakage, both of which can significantly affect intestinal function. These limitations highlight the need for more objective and quantitative approaches to evaluate protozoan-induced intestinal damage in turkeys.
Evans Blue Dye (EBD) is a well-characterized dye that binds strongly to plasma albumin and serves as a marker for evaluating vascular leakage and mucosal injury (Radu and Chernoff, 2013). In normal physiological conditions, albumin-bound EBD remains within the bloodstream, but when vascular or epithelial barriers are compromised, the dye escapes into surrounding tissues, providing a quantifiable measure of damage (Radu and Chernoff, 2013). Although this technique is widely employed in mammalian models (Goldim et al., 2019) to study vascular integrity and inflammation, its use in avian systems is limited. The current research note presents a straightforward and reproducible application of EBD to quantify intestinal damage in turkey poults infected with Eimeria spp. or Histomonas meleagridis, offering a more objective and quantitative alternative measurement to conventional lesion scoring methods.
Materials and methods
All animal experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Georgia under Animal Use Protocols (A202304-013 and A2024 06-021).
Experimental design and growth parameters
A total of 180 d-old male turkey poults were randomly assigned to three treatment groups, with five replicates and twelve poults per cage and kept for 28 d in battery cages. The groups included: NC (non-challenged control), EM (orally challenged on d 22 with 8,000 oocysts of Eimeria meleagrimitis and Eimeria adenoeides), and HM (cloacally challenged on d 18 with 100,000 trophozoites of Histomonas meleagridis). Eimeria oocysts were obtained by administering an over supplemented dose of Immucox® T turkey coccidiosis vaccine (Ceva, Libourne, France), providing 4,000 E. meleagrimitis and 4,000 E. adenoeides oocysts per bird. Histomonas meleagridis trophozoites were derived from a previously frozen culture (Buford strain), which was revived and propagated until reaching a concentration of 100,000 trophozoites, then used for the experimental challenge (Rafieian-Naeini et al., 2025a). On d 22, 25, and 28, body weight (BW) was recorded, and daily feed intake (DFI) was measured. Then, average weight gain (AWG) and feed conversion ratio (FCR) were calculated for the entire experimental period.
Preparation, injection of Evans Blue Dye and intestinal tissue collection
Evans Blue powder (Thermo Fisher Scientific Inc., Waltham, MA, USA) was used to prepare a 20 mg/mL solution in sterile 0.9% saline (Alkali Scientific, Fort Lauderdale, FL, USA). The resulting solution was filtered twice using sterile syringe filters (25 mm, 0.22 µm; VWR International, Radnor, PA, USA). On day 28, two birds per replicate (ten birds per treatment group) were selected. Each bird was slowly injected intravenously via the brachial vein using a 25-gauge needle with 10 mg/kg body weight of the prepared EBD solution, ensuring complete delivery of the dose. Birds were kept for one-hour post-injection and then euthanized by cervical dislocation. The neck was cut under running water to allow complete exsanguination. Following euthanasia, necropsy was performed, and approximately 2 g of tissue was collected from the liver, duodenum, jejunum, and ceca. To ensure consistency across birds, all samples were collected from standardized anatomical locations: the middle of the right liver lobe for liver tissue, and the same defined segment of the duodenum, jejunum, and ceca for each bird, regardless of lesion severity. Samples were rinsed gently with phosphate-buffered saline (PBS), placed into 2 mL microtubes, and frozen in liquid nitrogen. All samples were then transferred to the laboratory and stored at −20°C until further analysis.
Evans Blue Dye extraction and quantification
Collected tissue samples were dried in a 70°C oven for 48 hours. Then, 100 mg of the dried and finely ground tissue was mixed with 500 µL of formamide (Thermo Fisher Scientific Inc., Waltham, MA, USA) and incubated at 55°C for 24 hours. After incubation, the samples were centrifuged at 10,000 × g for 15 minutes. A 100 µL aliquot of the supernatant was transferred, in duplicate, into a microplate for absorbance measurement at 610 nm using a multimode microplate reader (Thermo Fisher Scientific Inc., Waltham, MA, USA). The Evans Blue concentration was determined using a standard curve generated from known EBD concentrations, and the results were expressed as nanograms (ng) of dye per milligram (mg) of dry tissue.
Lesion scores
At day 28, lesion scoring was performed for all birds following established criteria described in the literature. Ceca and liver lesions were evaluated for H. meleagridis (10dpi) following the methods in previous studies (0-4 scale) (Hauck et al., 2018; McDougald, 1997; Rafieian-Naeini et al., 2025a), while duodenal and cecal lesions were assessed for Eimeria meleagrimitis and Eimeria adenoeides (6dpi, 0-4 scale) based on the methods described by (Gadde et al. 2020).
Statistical analysis
Data were analyzed using JMP Pro 18 (SAS Institute Inc., Cary, NC) with a one-way ANOVA at a significance level of P ≤ 0.05. Group differences were further evaluated using Tukey’s post hoc test, and Student’s t-test was used for selected lesion-scoring comparisons.
Results and discussion
Growth data are presented in Table 1. At day 28, poults in the HM group exhibited the poorest growth performance, with lower BW (P = 0.004), FI (P = 0.005), and BWG (P < 0.01) compared to the NC and EM groups. The HM group also showed the highest FCR values during d 19–28 (P = 0.0004) and over the entire period (P = 0.008), indicating reduced feed efficiency. As previously described by Rafieian-Naeini et al. (2025a), anorexia and weight loss were observed around 4-5 days post-infection (dpi) as clinical signs of H. meleagridis infection in the current study. These symptoms are often associated with severe necrotic lesions in the ceca and liver, which compromise nutrient absorption and decrease the birds’ willingness to consume feed.
Table 1.
Body weight, feed intake, body weight gain, and feed conversion ratio of turkey poults challenged with Histomonas meleagridis and Eimeria spp.
| Parameter | Treatments1 |
Statistics |
|||
|---|---|---|---|---|---|
| NC | EM | HM | SEM2 | P value3 | |
| BW | |||||
| d 22 | 569.01 | 570.50 | 546.50 | 22.69 | 0.70 |
| d 25 | 692.07a | 674.83ab | 566.89b | 29.99 | 0.02 |
| d 28 | 786.54a | 718.41a | 575.39b | 36.60 | 0.004 |
| FI | |||||
| d 0-18 | 452.23 | 468.60 | 471.34 | 19.60 | 0.76 |
| d 19-28 | 538.25a | 448.38ab | 395.65b | 24.91 | 0.005 |
| d 0-28 | 990.49 | 916.98 | 866.99 | 34.28 | 0.072 |
| BWG | |||||
| d 0-18 | 363.00 | 373.88 | 374.69 | 21.48 | 0.91 |
| d 19-28 | 385.00a | 258.71b | 136.21c | 24.04 | <.0001 |
| d 0-28 | 748.01a | 632.59ab | 510.91b | 43.30 | 0.007 |
| FCR | |||||
| d 0-18 | 1.25 | 1.25 | 1.26 | 0.034 | 0.96 |
| d 19-28 | 1.41b | 1.79b | 2.98a | 0.20 | 0.0004 |
| d 0-28 | 1.33b | 1.46ab | 1.72a | 0.07 | 0.008 |
Within each row, means with different letters are significantly different (P value < 0.05).
A total of 180 d-old male turkey poults were allocated to three treatment groups (5 cages/group and 12 poults/cage). NC, NO challenge; EM, orally challenged with 8,000 oocysts of E. meleagrimitis and E. adenoeides on day 22; HM, cloacally challenged with 100,000 trophozoites of Histomonas meleagridis on day 18.
SEM: pooled standard error of means. N = 5.
P value: based on one-way ANOVA.
In contrast, poults challenged with Eimeria showed only moderate reductions in FI and growth in the current study, suggesting a partial recovery after the initial infection period. E. meleagrimitis primarily infects the duodenum and jejunum, whereas E. adenoeides targets the ceca, with visible lesions typically developing between 4 and 6 days post-infection (dpi) (Gadde et al., 2020; Rafieian-Naeini et al., 2025b). These lesions are characterized by intestinal inflammation and mucosal thickening, which transiently reduce feed intake and weight gain. Overall, EM-challenged poults showed a mild reduction in performance compared with those infected with H. meleagridis, while the NC group exhibited the best performance.
Lesion scores and EBD concentrations on d 28 (corresponding to 6 dpi for Eimeria and 10 dpi for H. meleagridis) are summarized in Table 2. The HM-challenged poults exhibited significantly higher liver lesion scores compared to the NC (P < 0.0001), consistent with the well-documented hepatic pathology caused by H. meleagridis (Fudge et al., 2024). Notably, EBD concentration in the liver of HM-infected birds was approximately 150% higher than that in non-challenged poults, indicating substantial vascular leakage and tissue damage caused by Histomonas challenge. As shown in Fig. 1, tissues with higher EBD accumulation appeared visibly darker blue, reflecting greater permeability.
Table 2.
Liver, intestinal lesion scores and Evans blue dye concentration in turkey poults challenged with Histomonas meleagridis and Eimeria spp.
| Parameter | Treatments1 |
Statistics |
|||
|---|---|---|---|---|---|
| NC | EM | HM | SEM2 | P value3 | |
| H. meleagridis lesion scoring system | |||||
| Liver | |||||
| Lesion score (0-4) | 0b | - | 2.25a | 0.17 | <.0001 |
| Evans blue (ng/mg) | 182.16b | - | 541.71a | 43.07 | <.0001 |
| Ceca | |||||
| Lesion score (0-4) | 0b | - | 2.85a | 0.18 | <.0001 |
| Evans blue (ng/mg) | 282.45b | - | 552.08a | 52.56 | 0.004 |
| Eimeria lesion scoring system | |||||
| Ceca | |||||
| Lesion score (0-4) | 0 | 0.72 | - | 0.11 | 0.34 |
| Evans blue (ng/mg) | 282.45 | 365.85 | - | 48.46 | 0.25 |
| Duodenum | |||||
| Lesion score (0-4) | 0b | 1.36a | - | 0.06 | <.0001 |
| Evans blue (ng/mg) | 134.10b | 232.53a | - | 29.03 | 0.04 |
| Jejunum | |||||
| Lesion score (0-4) | 0b | 0.36a | - | 0.04 | 0.05 |
| Evans blue (ng/mg) | 54.78b | 121.54a | - | 8.09 | 0.0004 |
Within each row, means with different letters are significantly different (P value < 0.05).
A total of 180 d-old male turkey poults were allocated to three treatment groups (5 cages/group and 12 poults/cage). NC, NO challenge; EM, orally challenged with 8,000 oocysts of E. meleagrimitis and E. adenoeides on day 22; HM, cloacally challenged with 100,000 trophozoites of Histomonas meleagridis on day 18.
For lesion scoring and EBD outcomes, statistical comparisons were performed within challenge model only: NC vs HM for liver and ceca (H. meleagridis scoring system) and NC vs EM for duodenum and jejunum (Eimeria scoring system). The non-relevant challenge group is shown for reference only and was not included in the statistical analysis for that tissue/scoring system.
SEM: pooled standard error of means. N = 10.
P value: based on one-way ANOVA.
Fig. 1.
Photographs of the liver, ceca, duodenum, and jejunum of turkey poults challenged with turkey poults challenged with Histomonas meleagridis and Eimeria spp., following injection with Evans blue dye (10 mg/kg BW) one hour prior to euthanasia and tissue collection.
Note: A total of 180 d-old male turkey poults were allocated to three treatment groups (5 cages/group and 12 poults/cage). NC, NO challenge; EM, orally challenged with 8,000 oocysts of E. meleagrimitis and E. adenoeides on day 22; HM, cloacally challenged with 100,000 trophozoites of Histomonas meleagridis on day 18.
Similarly, cecal lesion scores were markedly elevated in the HM group, with corresponding increases in EBD concentration, confirming the severe cecal damage typical of histomoniasis. In the EM group, moderate cecal lesions and a noticeable increase in EBD concentration were also observed, indicating mild to moderate intestinal damage consistent with Eimeria infection. In contrast, poults challenged with Eimeria showed significantly higher lesion scores in the duodenum and jejunum, the primary sites of E. meleagrimitis invasion, compared to the NC group, accompanied by elevated EBD levels in these intestinal regions. These findings align with previous observations that Eimeria infection induces localized intestinal inflammation and increased vascular permeability (Rafieian-Naeini et al., 2025b). Moreover, EBD has been measured in the muscle, liver, ileum, jejunum, spleen, and kidney, with increased EBD concentrations reported in heat-stressed broilers, particularly in the kidney and ileum, reflecting stress-induced compromise of tissue integrity. Dietary betaine has been shown to mitigate heat stress and reduce EBD accumulation in the kidney (Shakeri et al., 2019).
The findings of the present study demonstrate that Evans Blue Dye (EBD) serves as a promising and objective marker for assessing lesion severity in turkey poults infected with Eimeria and H. meleagridis. The use of EBD provides a quantitative tool to complement traditional subjective lesion scoring, helping to minimize observer bias and allowing detection of even mild or microscopic lesions that may otherwise go unnoticed. Its strong tissue infiltration and correlation with vascular damage make it a valuable measurement of tissue integrity. Overall, EBD provides a reliable and practical method for enhancing lesion evaluation and treatment assessment in poultry disease research. Future investigations should aim to further validate and standardize this approach across different pathogens, tissues, and experimental conditions.
Funding
This research did not receive any specific grant.
CRediT authorship contribution statement
Hamid Reza Rafieian-Naeini: Writing – original draft, Visualization, Validation, Methodology, Investigation, Formal analysis, Conceptualization. Woo Kyun Kim: Writing – review & editing, Supervision, Resources, Project administration, Investigation, Funding acquisition, Data curation, Conceptualization.
Disclosures
There is no conflict of interest.
Acknowledgement
The authors would like to thank Dr. Majid Shakeri and Dr. Brian Bowker for their valuable technical assistance and guidance as part of the research conducted at the U.S. National Poultry Research Center, USDA-ARS, Athens, GA, USA.
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