Integrated assessment of performance, hematology, and intestinal health in broilers under mixed Eimeria spp. challenge

Avaliação integrada do desempenho produtivo, parâmetros hematológicos e integridade intestinal de frangos de corte submetidos ao desafio por Eimeria spp.

ABSTRACT

Coccidiosis is a major enteric disease in poultry production, particularly under mixed infections involving multiple Eimeria species. This study evaluated the effects of a moderate mixed Eimeria spp. challenge on growth performance, hematological profile, and intestinal integrity in broiler chickens. A total of 288 broilers were allocated to challenged or unchallenged groups. At 14 days of age, challenged birds were orally inoculated with Eimeria tenella (3.5 × 10³ oocysts/bird), Eimeria acervulina (7.0 × 10⁴ oocysts/bird), and Eimeria maxima (1.0 × 10⁵ oocysts/bird). Performance was evaluated until 42 days of age, and samples were collected at 7 and 14 days post-challenge. Challenged birds showed reduced body weight gain, feed intake, and feed efficiency during the first week post-infection, along with increased mortality, higher oocyst shedding, and more severe intestinal lesions. Although oocyst excretion and lesion severity decreased by 14 days post-challenge, total intestinal health index scores remained elevated. Hematocrit decreased at 7 days post-challenge, while leukocyte count and heterophil-tolymphocyte ratio increased at 14 days post-challenge. These results indicate that mixed Eimeria infection compromises both intestinal integrity and systemic physiological responses, and that intestinal morphological recovery does not necessarily reflect complete systemic restoration.

Keywords
chicken; coccidiosis; intestinal health; hematological response; growth performance

RESUMO

A coccidiose é uma importante enfermidade entérica na produção avícola, especialmente sob infecções mistas por diferentes espécies de Eimeria. Este estudo avaliou os efeitos de um desafio misto moderado por Eimeria spp. sobre o desempenho produtivo, perfil hematológico e integridade intestinal de frangos de corte. Foram utilizadas 288 aves, distribuídas em grupos desafiados e não desafiados. Aos 14 dias de idade, as aves desafiadas foram inoculadas por via oral com Eimeria tenella (3,5 × 10³ oocistos/ave), Eimeria acervulina (7,0 × 10⁴ oocistos/ave) e Eimeria maxima (1,0 × 10⁵ oocistos/ave). O desempenho foi avaliado até 42 dias, com coletas aos 7 e 14 dias após o desafio. As aves desafiadas apresentaram redução no ganho de peso, no consumo de ração e eficiência alimentar na primeira semana pós-infecção, além de maior mortalidade, excreção de oocistos e lesões intestinais. Aos 14 dias pós-desafio, observou-se redução na excreção de oocistos e na severidade das lesões; contudo, o índice de saúde intestinal permaneceu elevado. Houve redução do hematócrito aos 7 dias e aumento da contagem de leucócitos e da razão heterófilo/linfócito aos 14 dias após o desafio moderado. Conclui-se que a infecção mista compromete a saúde intestinal e sistêmica e que a recuperação morfológica não indica necessariamente a restauração fisiológica completa.

Palavras-chave
frangos de corte; coccidiose; saúde intestinal; hematologia; desempenho produtivo

1. Introduction

Avian coccidiosis is one of the most impactful enteric diseases in global poultry production, causing significant impairment of intestinal health and substantial economic losses associated with reduced growth performance. It is estimated that, in 2022, global losses related to coccidiosis reached £12.9 billion (approximately €15 billion), including costs associated with prophylaxis, treatment, performance losses, and bird culling (Blake, 2025). Current control strategies rely on a combination of vaccination, prophylactic feeding, disinfectants, and management practices, with a strong dependence on anticoccidials (Zhao et al., 2024; Mathis et al., 2025; Cantin-Rosas et al., 2025). However, increasing regulatory restrictions and market demand for antibiotic-free production systems have limited the effectiveness of traditional control approaches, while the emergence of drug-resistant strains and variable vaccine responses further complicate disease management (Lillehoj et al., 2018).

Although severe clinical cases of coccidiosis are readily identifiable and treatable, subclinical infections represent a major challenge under commercial conditions. Evidence indicates that even low-intensity Eimeria spp. infections can impair zootechnical performance and predispose birds to secondary enteric disorders (Balestrin et al., 2021; Freitas et al., 2023). For example, Goo et al. (2023) demonstrated that increasing doses of Eimeria maxima, including subclinical levels, significantly reduced weight gain and feed efficiency, increased intestinal permeability, and promoted mucosal damage, thereby increasing susceptibility to necrotic enteritis.

The severity of coccidiosis depends on the infecting species, challenge dose, and age of the birds. While Eimeria acervulina and Eimeria maxima are primarily associated with reduced productivity, Eimeria tenella is linked to higher mortality and severe cecal lesions (Adhikari et al., 2020). Beyond productive losses, coccidiosis induces marked intestinal and hematological alterations, impairing nutrient absorption, disrupting epithelial integrity, and triggering inflammatory responses (Teng et al., 2020; Sharma et al., 2024). In mixed infections, histopathological damage may further exacerbate the risk of secondary infections, such as necrotic enteritis caused by Clostridium perfringens (Goo et al., 2023; Ke et al., 2024).

Importantly, intestinal morphological recovery does not necessarily indicate full physiological restoration. Although epithelial regeneration and reduction in parasite load may suggest resolution of the acute phase, systemic inflammatory activation may persist beyond apparent structural recovery (Teng et al., 2021). Persistent leukocyte alterations and elevated heterophil-to-lymphocyte ratios have been associated with prolonged immune activation and metabolic cost, potentially contributing to sustained performance impairment even after visible intestinal repair (Klasing, 2004; Cowieson et al., 2020)

Considering this pathophysiological framework, the characterization of the temporal progression of intestinal and systemic alterations in moderate mixed Eimeria spp. infection models are essential for an integrated understanding of host responses. In this context, the present study aimed to evaluate the evolution of intestinal morphological alterations and hematological parameters in broiler chickens subjected to a controlled mixed challenge, investigating the potential dissociation between structural restoration of the intestinal epithelium and resolution of systemic inflammatory activation.

2. Material and methods

2.1. Ethical approval

All experimental procedures were approved by the Animal Use Ethics Committee of the Federal University of Rio Grande do Sul (CEUA/UFRGS), under protocol number 35670, and were conducted in accordance with national and international guidelines for animal experimentation. Birds were monitored daily for health and welfare.

2.2. Experimental design and bird management

A total of 288 one-day-old Cobb broilers were reared from 1 to 42 days of age. Birds were assigned to two treatments: challenged and unchallenged, with eight replicate pens per treatment (16 pens total). Each floor pen (1 m²) was considered the experimental unit for performance analysis.

Birds were housed in two environmentally controlled rooms under identical management, temperature, ventilation, lighting, and biosecurity conditions. Challenged and unchallenged groups were allocated in separate rooms to prevent cross-contamination. Biosafety procedures included footbaths, clothing changes, disposable caps and shoes, and sequential handling.

Stocking density was 18 birds/m² from 1 to 21 days of age and reduced to 12 birds/m² from 22 to 42 days.

Birds were weighed weekly, and feed intake was calculated as the difference between feed offered and feed refusal. Average daily gain (ADG), average daily feed intake (ADFI), and feed conversion ratio (FCR) were calculated. Mortality was recorded daily and used to adjust performance variables.

At 21 and 28 days of age (7 and 14 days post-challenge), litter and blood samples were collected. Three birds per pen were euthanized by cervical dislocation for macroscopic and histological evaluation.

2.3. Diets

Three diets were provided throughout the experimental period: pre-starter (1–7 days), starter (8–21 days), and finisher (22–42 days). Diets were formulated according to the Brazilian Tables for Poultry and Swine (Rostagno et al., 2017) (Table 1).

Table 1
Ingredient formula and chemical composition of experimental diets according to the rearing period.

	Pre-starter (1 – 7 days)	Starter (8 – 21 days)	Finisher (22 – 42 days)
Formula (%)
Corn 7.8% CP	53.55	57.27	60.13
Soybean meal 45% CP	38.82	35.15	31.86
Soybean oil	2.94	3.37	4.29
Dicalcium phosphate	1.93	1.72	1.48
Calcitic limestone	0.94	0.87	0.74
Sodium chloride	0.53	0.52	0.49
DL-methionine	0.38	0.34	0.33
L-lysine	0.31	0.26	0.22
L-threonine	0.16	0.08	0.08
Mineral premix1	0.10	0.10	0.07
Vitamin premix2	0.05	0.05	0.05
Antifungal	0.10	0.10	0.10
Acidifying	0.10	0.10	0.10
Mycotoxin adsorbents	0.07	0.07	0.07
Antioxidant	0.01	0.01	0.01
Chemical composition (%)
Metabolizable Energy (EM/kg)	2975	3050	3150
Crude protein	22.45	21	19.69
Total lysine	1.45	1.384	1.29
Digestible Lysine	1.33	1.27	1.18
Total Methionine+Cystine	0.87	0.82	0.75
Digestible Methionine+Cystine	0.67	0.71	0.62
Total threonine	1.00	0.88	0.82
Digestible thronine	0.89	0.78	0.73
Total tryptophan	0.28	0.26	0.24
Digestible tryptophan	0.25	0.23	0.22
Sodium	0.23	0.22	0.21
Chlorine	0.39	0.38	0.36
Potassium	0.88	0.83	0.78
Calcium	0.97	0.88	0.76
Phosphorus available	0.46	0.42	0.37
Total phosphorus	0.59	0.65	0.56
Crude fiber	2.81	2.70	2.59
Ethereal extract	5.73	6.23	7.19

• ¹
  Composition (per kg of product): 88.000 mg of Manganese, 95.535 mg of Zinc, 64.715 mg of Iron, 15.000 mg of Copper, 1.795 mg of Iodine, 200 mg of Selenium.
• ²
  34.520,000 UI of vitamin A, 7.200,000 UI of vitamin D, 8.600 mg of vitamin K, 6.700 mg of vitamin B1, 20.000 mg of vitamin B2, 9.000 mg of vitamin B6, 132.000 mg of niacin, 34,000 mg of pantothenic acid, 2.800 mg of folic acid, 240 mg of biotin, 72.000 μg of vitamin B12.

2.4. Experimental challenge

At 14 days of age, birds in the challenged group were orally inoculated with 1.0 mL containing sporulated oocysts of Eimeria tenella (3.5 × 10³ oocysts/bird), Eimeria acervulina (7.0 × 10⁴ oocysts/bird), and Eimeria maxima (1.0 × 10⁵ oocysts/bird), obtained from the CAPEV laboratory.

The selected doses were designed to induce a controlled moderate mixed infection model. Birds were fasted for 3–4 hours prior to inoculation, with free access to water. The inoculum was administered orally using a syringe fitted with a probe.

Birds in the unchallenged group received 1.0 mL of sterile saline solution via the same oral gavage procedure.

2.5. Oocyst quantification

Oocyst quantification was performed according to Costa and Paiva (2009) using a McMaster chamber. Litter samples were collected from five different locations within each pen, homogenized, and analyzed at 7 and 14 days post-inoculation. Species identification was performed by morphometric evaluation, and the total oocysts per pen were calculated as:

$Oocysts/pen=\frac{oocysts\times dilutionfactor\times samplevolume}{chambervolume}$

2.6. Hematological analysis

At 7 and 14 days post-challenge, blood samples were collected from the ulnar vein of one bird per pen prior to euthanasia. The same birds were subsequently used for histopathological evaluation of the intestine. The following parameters were evaluated: hematocrit, hemoglobin, total leukocyte count, differential leukocyte count (heterophils, lymphocytes, eosinophils, basophils, and monocytes), and heterophil-to-lymphocyte ratio.

2.7. Intestinal lesion scoring

At 21 and 28 days of age (7 and 14 days postchallenge), three birds per pen were evaluated for macroscopic intestinal lesions in the duodenum, jejunum, ileum, and cecum, according to the scoring system described by Johnson and Reid (1970), ranging from 0 (no lesions) to 4 (severe lesions).

2.8. Histopathological analysis

Jejunal samples were collected in a standardized manner from the mid-portion of the jejunum, fixed in 10% buffered formalin, routinely processed, and embedded in paraffin. Sections (5 µm) were stained with hematoxylin and eosin and alcian blue. Twenty well-oriented villi per bird were evaluated using a light microscope (Leica DM1000 LED).

Histopathological evaluations were performed by a single trained evaluator blinded to treatment allocation in order to minimize observational bias.

Intestinal health was assessed using the Intestinal Health Index (ISI) methodology (Kraieski et al., 2017), calculated as:

$𝐼𝑆𝐼=𝛴(𝑙𝑒𝑠𝑖𝑜𝑛𝑠𝑐𝑜𝑟𝑒\times 𝑖𝑚𝑝𝑎𝑐𝑡𝑓𝑎𝑐𝑡𝑜𝑟)$

Parameters evaluated included lamina propria thickness, epithelial thickness, enterocyte proliferation, inflammatory cell infiltration (epithelium and lamina propria), goblet cell increase, congestion, and presence of Eimeria oocysts (Table 2).

Table 2
ISI histological alterations evaluated.

Alteration	Impact factor
Lamina propria thickness	2
Epithelial thickness	1
Enterocystes proliferation	1
Inflammatory cell infiltration in the epithelium	1
Inflammatory cell infiltration in the lamina propria	3
Geblet cells	2
Congestion	2
Presence of oocysts	2
Maximum score1	45

• ¹
  Maximum score represents the sum of all alterations according to the formula ISI = Σ (IS×IF), where IS = lesion score (0-3) (observed) and IF = impact factor (1-3) (preestablished).

2.9 Statistical analysis

Data were analyzed separately by age or experimental period, considering the pen as the experimental unit. Normality and homogeneity of variances were assessed using the Shapiro–Wilk and Levene tests, respectively.

When assumptions were met, treatment means were compared using Student’s t-test for independent samples. Oocyst data were analyzed using the Wilcoxon rank-sum test when distributional assumptions were not satisfied.

Statistical significance was set at p ≤ 0.05. Results are presented as means ± standard error of the mean (SEM).

3. Results and discussion

Mixed infections with Eimeria acervulina, Eimeria maxima, and Eimeria tenella are frequently observed in commercial poultry production systems, where moderate infections are more common than severe experimental outbreaks. In such scenarios, productive losses often occur in the absence of evident clinical signs, making the integrated evaluation of intestinal and systemic responses essential. In the present study, the productive, parasitological, hematological, and intestinal responses of broilers subjected to a controlled moderate mixed Eimeria spp. challenge were evaluated. Results are presented according to the evaluation period and discussed in the context of infection dynamics, highlighting the relationship between acute intestinal damage, subsequent partial tissue recovery, and the persistence of systemic inflammatory indicators.

3.1. Growth performance, oocyst shedding, and intestinal lesions following Eimeria spp. challenge

Body weight did not differ between groups at 14 days of age (p > 0.05), confirming homogeneous baseline conditions prior to inoculation (Table 3). However, from 21 days onward, challenged birds exhibited significantly lower body weight (p < 0.01), and this difference persisted until 42 days of age, indicating a sustained negative impact of the mixed Eimeria infection on growth performance. This reduction corresponded to a 23.7% decrease in body weight at 21 days of age, reflecting the marked impact of the acute phase of infection. Although the magnitude of the difference decreased over time, challenged birds still exhibited reductions of 15.8% and 9.7% at 28 and 42 days of age, respectively, demonstrating that early intestinal damage resulted in persistent impairment of growth performance.

Table 3
Evaluation of poultry performance, body weight (BW), daily body weight gain (BWG), daily feed intake (FI), feed conversion (FCR), and mortality (MORT) in a mixed challenge model with Eimeria spp.

Parameters	Age (days)	Challenged	Unchallenged	p-value	SEM
BW (g)	14	490	479	0.307	7.89
	21	745	977	0.000	13.25
	28	1387	1648	0.000	25.52
	42	3034	3360	0.002	46.01
BWG (g/day)	1-14	31	31	0.311	0.56
	14-21	36	71	0.000	2.53
	21-28	92	96	0.334	4.91
	28-42	117	122	0.361	2.52
	1-42	71	79	0.002	3.56
FI (g/day)	1-14	39	38	0.359	3.65
	14-21	72	93	0.000	3.34
	21-28	134	145	0.043	4.71
	28-42	191	193	0.566	2.89
	1-42	111	117	0.013	4.25
FCR (g/g)	1-14	1.23	1.23	0.930	2.53
	14-21	2.00	1.32	0.000	0.04
	21-28	1.47	1.52	0.360	0.24
	28-42	1.62	1.58	0.313	0.03
	1-42	1.56	1.48	0.005	0.25
MORT (%)	1-14	0.42	0.56	0.559	0.04
	14-21	6.95	0.43	0.035	1.09
	21-28	0.96	0.24	0.334	0.25
	28-42	0.76	0.80	0.952	0.23
	1-42	8.08	3.39	0.025	0.36

• Data are presented as means (pen as experimental unit; n = 8). Means were compared using Student’s t-test (p < 0.05). SEM = standard error of the mean.

The greatest impairment occurred during the acute phase (14–21 days), when challenged birds showed reduced body weight gain, decreased feed intake, increased feed conversion ratio (p < 0.001), and higher mortality (p < 0.05). A total of 17 birds died during the experimental period, corresponding to 12 birds in the challenged group and 5 in the unchallenged group, based on mortality percentages and initial stocking density. Most mortality occurred during the acute phase (14–21 days), coinciding with peak oocyst shedding and lesion severity. Although dead birds were not subjected to necropsy, affected animals showed clinical signs consistent with coccidiosis, including reduced activity, ruffled feathers, decreased feed intake, and, in some cases, bloody droppings, which are commonly associated with Eimeria tenella infection. These observations support the association between mortality and the experimental challenge. This period coincided with peak oocyst shedding and greater lesion severity (Figure 1), supporting the relationship between parasite replication, intestinal tissue damage, and productive losses (Kraieski et al., 2017; Teng et al., 2020). No blood or other laboratory analyses were performed on birds that died during the experimental period. Performance variables, including feed intake and body weight gain, were adjusted for mortality at the pen level, ensuring accurate estimation of productive parameters. Therefore, hematological and histopathological results reflect only the surviving birds evaluated at predefined sampling time points.

Figure 1
Species-specific oocyst shedding and intestinal lesion scores at 7 and 14 days post-challenge in broilers experimentally infected with mixed Eimeria spp. The bars represent the mean number of oocysts shed, and the lines the lesion score caused by each Eimeria spp. Letters indicate significant differences (p < 0.01). Error bars represent standard error (SE) values. Birds were challenged with Eimeria spp. at 14 days of age. The challenge dose was 3.5 × 10³ E. tenella oocysts, 7.0 × 10⁴ E. acervulina oocysts, and 1.0 × 10⁵ E. maxima oocysts.

Oocyst shedding was markedly higher at 21 days of age (7 days post-challenge) and decreased substantially by 28 days (14 days post-challenge), characterizing a typical moderate infection profile. An average reduction of 88% in oocyst excretion was observed between these time points, indicating progressive immune control of parasite replication. Importantly, lesion scores and mortality levels remained within ranges commonly associated with moderate field infections rather than severe experimental models, reinforcing the relevance of this challenge design to commercial production conditions (Cornelissen et al., 2009; Adhikari et al., 2020; Goo et al., 2023).

Among species, E. tenella showed the highest oocyst counts and lesion scores, which may explain the observed severity of cecal lesions, given its recognized pathogenic potential and association with hemorrhagic damage (Choi et al., 2021; Xu et al., 2022).

Although lesion severity decreased at 28 days, scores did not return to baseline levels. The temporal coincidence between peak parasitism, intestinal damage, and the most pronounced productive impairment reinforces the direct biological association between epithelial disruption, inflammatory response, and reduced growth efficiency (Sharma et al., 2024; Goo et al., 2023). Moreover, despite the reduction in parasite burden after the acute phase, cumulative performance remained compromised, suggesting that early mucosal injury imposed lasting productive consequences, an outcome frequently observed under moderate subclinical to clinical infections in commercial poultry systems.

3.2. Hematological responses under moderate mixed Eimeria infection

Hematological parameters were significantly affected by the mixed Eimeria challenge (Table 4), indicating that the infection extended beyond the intestinal compartment and elicited systemic physiological responses.

Table 4
Hematological indices on days 21 (7 days post-challenge) and 28 (14 days post-challenge) in broilers challenged and unchallenged with mixed coccidiosis.

Parameters	Age (days)	Challenged	Unchallenged	p-value	SEM
Hematocrit (%)	21	26.00	30.25	0.045	1.850
	28	31.00	30.00	0.175	0.756
Leukocytes (×103/µL)	21	29.91	27.14	0.594	4.160
	28	27.35	18.41	0.045	2.750
Heterophils (%)	21	51.63	32.88	0.043	4.200
	28	58.25	37.38	0.048	3.140
Lymphocytes (%)	21	42.63	30.25	0.036	5.510
	28	40.75	31.25	0.085	3.200
Monocytes (%)	21	7.00	15.74	0.811	5.490
	28	17.88	16.38	0.854	0.453
H/L ratio1	21	0.81	0.83	0.853	0.102
	28	1.62	0.95	0.022	0.025
Eosinophils (%)	21	7.88	7.75	0.975	3.310
	28	7.62	7.25	0.442	0.778

• Data are presented as means (n = 8 per treatment and age). Means were compared by Student’s t-test (p < 0.05). SEM = standard error of the mean; H/L ratio = heterophil-to-lymphocyte ratio.

Hematological analysis demonstrated that the infection extended beyond localized intestinal damage and elicited systemic physiological responses. At 7 days post-challenge, challenged birds showed reduced hematocrit values, likely reflecting transient anemia associated with intestinal hemorrhage and impaired nutrient absorption during peak mucosal disruption (Natt & Herrick, 1955; Cowieson et al., 2020). Concurrently, increased lymphocyte percentages suggest activation of adaptive immune mechanisms in response to parasitic antigens (Klasing, 2004)

By 14 days post-challenge, hematocrit values approached those of the unchallenged group, indicating partial physiological stabilization. However, total leukocyte count, heterophils, and the heterophilto-lymphocyte (H/L) ratio were significantly elevated. The H/L ratio is widely recognized as a biomarker of systemic inflammatory stress and prolonged immune activation. The persistence of leukocytosis and elevated H/L ratio despite the marked reduction in oocyst shedding indicates that systemic immune activation extended beyond the period of maximal parasite replication.

This pattern is particularly relevant in moderate infections, typically observed in commercial settings, where visible clinical signs may subside while inflammatory and metabolic costs persist. Persistent immune activation has been associated with increased nutrient partitioning toward immune function rather than growth, contributing to sustained impairment in growth performance even after apparent intestinal recovery (Klasing, 2004; Cowieson et al., 2020).

3.3. Histopathological alterations and partial structural recovery

Histopathological evaluation confirmed that the mixed Eimeria challenge induced marked intestinal alterations during the acute phase of infection (Table 5). At 21 days of age (7 days post-challenge), challenged birds exhibited significantly increased lamina propria thickness (p < 0.001), pronounced inflammatory infiltration in both the epithelium and lamina propria (p < 0.05), reduced epithelial thickness (p < 0.05), decreased goblet cell counts (p < 0.001), and marked vascular congestion (p < 0.001). The presence of oocysts was intense during this period, confirming active parasite replication.

Table 5
Intestinal Health Index histological alteration scores (ISI methods) evaluated in the jejunum at 21 and 28 days of age, associated with the coccidiosis mix.

Organ alteration	Age (days)	Challenged	Unchallenged	p-value	SEM
Lamina propria thickness	21	2.63	1.32	0.000	0.096
	28	2.31	1.40	0.045	0.099
Epithelial thickness	21	1.00	1.08	0.021	0.025
	28	1.11	1.09	0.723	0.031
Enterocyte proliferation	21	1.00	0.97	0.317	0.024
	28	0.98	0.98	0.221	0.014
Inflammatory infiltration of the epithelium
	21	0.60	0.10	0.040	0.000
	28	0.00	0.01	0.761	0.008
Inflammatory infiltration of the Lamina propria
	21	0.13	0.00	0.000	0.000
	28	0.22	0.17	0.000	0.055
Globet cells	21	1.80	2.02	0.000	0.017
	28	1.93	1.98	0.463	0.044
Congestion	21	1.93	1.33	0.000	0.096
	28	1.98	1.63	0.004	0.085
Presence of Oocysts	21	3.90	0.00	0.000	0.000
	28	1.40	0.00	0.567	0.260
Total	21	12.40	6.72	0.000	0.168
	28	9.93	8.52	0.002	0.402

• Data are presented as means (n = 24 birds per treatment and age; three birds per pen). Means were compared by Student’s ttest (p < 0.05). ISI = Intestinal Health Index (Σ IS × IF); SEM = standard error of the mean.

The total ISI score was significantly higher in challenged birds compared to unchallenged controls (12.40 vs. 6.72; p < 0.001), indicating substantial compromise of intestinal integrity. These alterations coincide with peak oocyst shedding and the most pronounced productive impairment, reinforcing the biological link between parasite replication, mucosal disruption, and growth depression (Kraieski et al., 2017; Teng et al., 2020).

At 28 days of age (14 days post-challenge), most structural parameters showed attenuation. Epithelial thickness and goblet cell numbers approached control values, and inflammatory infiltration in the epithelium was no longer significant. However, lamina propria thickness and vascular congestion remained elevated (p < 0.05), and the total ISI score was still significantly higher in challenged birds (9.93 vs. 8.52; p = 0.002).

These findings demonstrate partial mucosal recovery following a reduction in parasite burden. Importantly, lesion severity and ISI values remained within ranges typically associated with moderate field infections rather than severe experimental models, supporting the relevance of this challenge design to commercial poultry conditions.

The persistence of structural alterations, even after a substantial reduction in oocyst shedding, suggests that tissue repair lags behind parasite clearance. Such delayed recovery may contribute to the sustained productive impairment observed throughout the production cycle (Chen et al., 2025).

From a practical perspective, the present findings suggest that moderate mixed Eimeria infections, commonly encountered under commercial production systems, may exert longer-lasting productive and physiological effects than those inferred solely from lesion severity or oocyst excretion patterns (Graham et al., 2023). The dissociation observed between partial intestinal morphological recovery and persistent systemic inflammatory activation indicates that visible attenuation of lesions does not necessarily reflect complete physiological stabilization.

Therefore, exclusive reliance on macroscopic lesion scoring or fecal oocyst counts may underestimate the underlying immune-metabolic burden associated with moderate coccidiosis. Integrating growth performance data with systemic indicators such as hematocrit and heterophil-to-lymphocyte ratio may contribute to a more comprehensive interpretation of flock health status. These findings highlight the importance of considering moderate coccidiosis not only as an acute enteric disturbance but as a condition capable of inducing prolonged adaptive responses that may influence overall production efficiency.

4. Conclusions

Moderate mixed Eimeria spp. infection impairs growth performance and induces marked intestinal and hematological alterations in broiler chickens. Although intestinal lesions decrease over time, systemic inflammatory activation persists beyond partial mucosal recovery. These findings reinforce the importance of integrated evaluation strategies for accurate assessment of coccidiosis under production conditions.

Acknowledgments

The authors acknowledge the financial support provided by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) through the Universal Call (Process No. 407959/2023-8), and by the Fundação de Amparo à Pesquisa e Inovação do Estado de Santa Catarina (FAPESC), under Public Call No. 20/2024.

• Declaration of generative AI and AI-assisted technologies in the writing process
  During the preparation of this work, the author(s) used ChatGPT (OpenAI) to verify the English language and improve grammatical accuracy. After using this tool, the author(s) reviewed and edited the content as needed and take full responsibility for the content of the publication.

Data Availability

Data will be made available on request.

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Edited by

• Editor:
  Mario Celso Sperotto Brum

Publication Dates

• Publication in this collection
  07 Sept 2026
• Date of issue
  2026

History

• Received
  02 Mar 2026
• Accepted
  16 June 2026