Attenuated Vaccine Associated with Organic Acids in the Control of Coccidiosis and Promotion of Intestinal Health in Broiler Chickens

ABSTRACT

This study evaluated the efficacy of an attenuated coccidiosis vaccine containing early-maturing Eimeria strains, combined with a nutritional additive, as an alternative to anticoccidial drugs in broilers. A total of 672 male Ross chicks were allocated in a 4×2 factorial design (4 control programs: anticoccidial, additive + anticoccidial, vaccine and additive + vaccine vs. challenged or not with live coccidiosis vaccine and Escherichia coli), totaling eight treatments with six replicates of 14 birds. Data were analyzed using ANOVA (GLM procedure, SAS software). Challenged birds showed reduced productive performance (p<0.05) regardless of treatment, with no differences between control programs. The vaccine improved jejunal morphometry, notably villus width and crypt depth, and was associated with enhanced regenerative capacity at 28 days. No significant effects (p>0.05) were observed for mitotic index. Serum FITC-d levels were lower (p<0.05) in challenged birds, and foot coloration was higher with the vaccine plus additive. These findings support the use of an early attenuated vaccine as a viable and effective alternative to traditional anticoccidials, especially when paired with nutritional additives.

Keywords:
Attenuated vaccine; broiler chicken; coccidiosis; gut health; zootechnical performance

INTRODUCTION

Global food demand, especially for protein, is expected to increase sharply in the coming decades due to global population growth (estimated to reach approximately 10 billion by 2050), socioeconomic changes such as increased urbanization and higher incomes in developing countries, as well as a greater appreciation for the importance of high-quality protein in a healthy life (FAO, 2009; Mottet & Tempio, 2017). Among the main types of meat produced worldwide, poultry has shown the highest absolute and relative growth rate over the past 50 years (Windhorst, 2017). This trend has been driven by the greater accessibility of chicken meat compared to red meats, its associated health benefits, and religious and cultural considerations (Baldi et al., 2020).

However, this growth requires continuous advances in technology, genetics, nutrition, and animal welfare to ensure efficient large-scale production (Rosalen et al., 2020). Among the tools used for this purpose, antibiotic growth promoters (AGPs) were widely employed to maintain intestinal health and zootechnical performance in poultry. Nevertheless, prolonged use of these agents contributed to the development of bacterial resistance, leading the European Union to ban their use as performance-enhancing additives (Huyghebaert et al., 2011), a measure that has been followed by several other countries (Yang et al., 2020).

The prohibition and withdrawal of AGPs were prompted by concerns about the emergence of antibiotic-resistant bacteria, since over time and under certain conditions, some bacteria are capable of transferring resistance genes to one another, which can and reduce the effectiveness of treatment in both animals and humans (Yang et al., 2020).

In this context, the search for effective alternatives to AGPs becomes essential, particularly in the control of enteric diseases such as avian coccidiosis, one of the main diseases affecting broiler production. Infection by the protozoa of the genus Eimeria compromises intestinal integrity, reduces feed conversion, and increases production costs (Pinheiro et al., 2014). Its life cycle includes two phases: an endogenous phase, where asexual and sexual replication stages occur, causing destruction of intestinal epithelial cells and resulting in the release of immature oocysts into the environment; and an exogenous phase, during which oocysts sporulate in the environment to become infective again (Conway & McKenzie, 2007).

Beyond the direct damage, coccidiosis is an important predisposing factor for necrotic enteritis, which is caused by the excessive growth of Clostridium perfringens and its toxin production (Assis et al., 2010). This leads to serum leakage into the intestine, which stimulates mucus production and provides a richer nutrient source for C. perfringens proliferation (Forder et al., 2012). Thus, coccidiosis not only affects poultry health but also compromises food safety (Cosby et al., 2015).

The primary method used in the industry to control the disease is the addition of chemical or ionophore anticoccidials to the feed. However, prolonged use of these drugs has led to the emergence of resistant strains (Quiroz-Castañeda & Dantán-González, 2015). In addition, growing consumer concerns and increasing regulations regarding the potential ban of anticoccidials as feed additives have driven the search for alternative strategies. Among these, vaccines stand out as the most effective option, as they not only successfully control the disease but also increase the sensitivity of Eimeria spp. to anticoccidial drugs (Peek & Landman, 2011).

The attenuation of vaccines by precocity involves selecting oocysts that complete their life cycle up to 30 hours earlier than parasites from the original strain. This process induces early immunity and significantly reduces the reproductive capacity of the protozoa (Innes & Vermeulen, 2006). Moreover, this technology preserves intestinal integrity and may indirectly prevent other diseases associated with coccidiosis (Ronsmans et al., 2015).

In addition, organic acids (OAs) have been used for decades by feed manufacturers to control pathogenic microorganisms through competitive exclusion, and as a potential alternative to antibiotic growth promoters. They improve nutrient utilization and, consequently, zootechnical performance (Adil, 2010). These naturally occurring compounds found in plants and animals help balance the intestinal microbiota, enhance gut morphology, and unlike antibiotics, do not induce microbial resistance (Menten et al., 2014).

Among the main organic acids used in animal nutrition are butyric acid and lauric acid, both of which have potent antimicrobial activity. These compounds promote the growth of beneficial bacteria and inhibit pathogenic microorganisms such as Salmonella spp., Escherichia coli, and Campylobacter jejuni in the digestive tract of poultry (Van Deun et al., 2008). Additionally, they help lower digesta pH, stimulate pancreatic secretion, and exert trophic effects on the gastrointestinal mucosa, thereby supporting intestinal integrity and improving productive performance (Dibner & Buttin, 2002).

Therefore, combining a precocity-attenuated vaccine with organic acid supplementation may represent a synergistic strategy for coccidiosis control, promoting gut health and zootechnical performance in poultry even in the absence of anticoccidials. The objective of this study was to evaluate the efficacy of this integrated approach as a viable and sustainable alternative for the control of coccidiosis in broiler chickens.

MATERIALS AND METHODS

Treatments and experimental design

The study was conducted in the experimental animal facility of the Federal University of Paraná (UFPR) - Palotina campus. All procedures involving animal rearing and biological sample collection were approved by the Ethics Committee on Animal Use in Experimentation under protocol CEUA 19/2020.

A total of 672 one-day-old male chicks from the Ross strain sourced from 40-week-old breeders were housed. The birds were distributed in a completely randomized 4×2 factorial design (4 control programs: anticoccidial, additive + anticoccidial, vaccine and additive + vaccine vs. challenged or not with live coccidiosis vaccine and Escherichia coli), totaling 8 treatments with 6 replicates of 14 birds each, totaling 48 experimental units. Birds that received the vaccine were immunized via spray at the hatchery with a live coccidiosis vaccine containing five precocity-attenuated Eimeria species (E. acervulina, E. maxima, E. mitis, E. praecox, and E. tenella). Meanwhile, the coccidiostat program consisted of 80 g/kg narasin and 80 g/kg nicarbazin included in the feed at a dosage of 0.5 kg/ton.

The additive diet included a commercial blend of short-chain (butyric acid) and medium-chain (lauric acid) fatty acids at 1.5 kg/ton of feed, as recommended by the manufacturer.

Birds were housed in cages placed in separate rooms, one for challenged and another for non-challenged groups. Each cage (100 cm × 80 cm) was lined with autoclaved shredded paper. Six cages were used per treatment. The environmental conditions were kept within the thermal comfort range recommended by the strain’s manual, with heating provided by electric heaters and air conditioners, and air circulation ensured by exhaust fans installed in the walls.

Water was supplied using manual drinkers (one per cage) during the first week of life. From the second week until 28 days of age, nipple drinkers (six per cage) were used. Water was cleaned and replenished three times daily to maintain quality and appropriate temperature.

Bird management followed a specific protocol to avoid cross-contamination between experimental groups. Birds from the control group were handled first, followed by those from the challenged group.

Experimental diets based on corn, soybean meal, and wheat bran were formulated to meet the nutritional requirements for the different growth phases. The nutritional program was divided into two phases: starter (1 to 14 days of age) and grower (15 to 28 days), according to recommendations from local poultry businesses (Table 1).

Table 1
Ingredients and nutritional composition of diets.

Item	Starter	Grower
Corn	309.60	346.60
Wheat bran	220.00	220.00
Soybean meal	390.00	348.00
Soybean Oil	42.00	50.00
Calcitic Limestone	10.40	9.20
Dicalcium Phosphate	10.00	9.00
Salt	4.70	4.20
L-Lysine (60%)	3.30	3.50
DL-Methionine (99%)	3.10	2.80
L-Threonine (99%)	1.00	1.00
L-Valine (99%)	1.40	1.40
Vitamin-mineral premix1	2.00	2.00
Choline Chloride (60%)	0.50	0.30
Kaolin2	2.00	2.00
Lucantin yellow (10%)	0.20	0.20
Nutritional levels
ME (kcal/kg)	2976.33	3076.08
Crude Protein (%)	23.05	21.49
Ethereal extract (%)	6.33	7.19
Crude Fiber (%)	4.00	3.78
Calcium (%)	0.87	0.79
Available Phosphorus %	0.43	0.41
Digestible lysine (%)	1.28	1.19
Digestible methionine (%)	0.62	0.57
Digestible Met + Cys (%)	0.95	0.88
Digestible tryptophan (%)	0.26	0.24
Digestible threonine (%)	0.84	0.79
Digestible arginine (%)	1.41	1.29

• ¹
  Vitamin premix content (per kg of premix): Vit. A: 4.000 IU; Vit. D3: 1.167 IU; Vit. E: 10.000 IU; Vit. K3: 1.000 mg; Vit. B1: 1.000 mg; Vit. B2: 2.666.67 mg; Vit. B6: 1.667.00 mg; Vit. B12: 6.666.67 µg; Niacin: 13.333.33 mg; Pantothenic acid: 6.000 mg; Folic acid: 833.33 mg; Biotin: 80.000 µg; Antioxidant: 22.200 mg. Mineral premix content (per kg of premix): Iron: 23.333.33 ppm; Copper: 2.666.67 ppm; Iodine: 333.3 ppm; Zinc: 33.333.33 ppm; Manganese: 40.000 ppm; Selenium: 80 ppm. Enzymes and other additives (per kg of premix): Phytase: 16.667 g/kg; Enzyme complex (xylanase, amylase, protease): 33.33 g/kg.
• ²
  Kaolin was replaced by the nutritional additive (1.5 kg/ton). or the anticoccidial product (0.5 kg/ton).

At 14 days of age, birds were challenged via inoculation with a live coccidiosis vaccine and Escherichia coli. Birds assigned to treatments 5, 6, 7, and 8 received a commercial live anticoccidial vaccine containing E. acervulina, E. maxima, E. praecox, E. tenella, and E. mitis. The vaccine was administered directly into the crop at a dose 20 times higher than the manufacturer’s recommended concentration. Two days following vaccination, the challenged birds received a 2 mL crop inoculation containing 10⁹ CFU/bird of Escherichia coli (ATCC® 8739™).

Growth performance

Productive performance parameters - including feed intake (g), body weight gain (g), and feed conversion ratio - were assessed weekly. Feed consumption was obtained by calculating the difference between feed supplied and feed remaining for each period, divided by the total number of birds in each replicate. Feed conversion ratio was adjusted for weekly mortality, following the methodology described by Sakomura & Rostagno (2007).

Intestinal Morphology

At 19 and 28 days of age, two birds per replicate - totaling 12 birds per treatment - were euthanized for the collection of intestinal samples. Segments approximately 5 cm in length from the jejunum were longitudinally opened, mounted on Styrofoam plates, and rinsed with physiological saline. The samples were then fixed in buffered formalin, embedded in paraffin, sectioned into semi-serial 5 μm slices, and stained with hematoxylin and eosin (H&E).

Morphometric analysis was conducted using light microscopy (10× objective), with image capture performed via the ImagePro-Plus software system (version 5.2 - Media Cybernetics). Measurements included the height and width of 20 villi and the depth and width of 20 crypts per section. The absorptive surface area (ASA) was calculated according to the formula proposed by Kisielinski et al. (2002)

$\begin{array}{l}ASA=\left(VWxVH\right)+{\left(VW/2+CW/2\right)}^2-{\left(VW/2\right)}^2\\ {\left(VW/2+CW/2\right)}^2\end{array}$

(LW: Villus Width, VH: Villus Height, CW: Crypt Width).

Assessment of Intestinal Permeability via Serum FITC-d Levels

Intestinal permeability was assessed by the oral administration of fluorescein isothiocyanate-dextran (FITC-d) and subsequent blood quantification. Five days after enteric challenge (19 days of age), FITC-d was administered orally to two birds per replicate (2.2 mg/mL, FITC-d, 100 mg, MW 4,000; Sigma-Aldrich). Birds were fasted for two hours before blood collection, and FITC-d concentration was determined per mL of serum. The presence of FITC-d in the bloodstream indicates increased intestinal permeability, as this molecule does not normally cross the intestinal barrier under normal mucosal integrity. However, in cases of enteric inflammation and disruption of cell junctions, FITC-d translocates into circulation and is detected in the analysis.

Immunohistochemical Assessment of Jejunal Cell Proliferation Using PCNA Marker

Cell proliferative activity was assessed using immunohistochemistry with the PCNA (Proliferating Cell Nuclear Antigen) marker. Previously embedded jejunal samples were sectioned into 5 μm-thick slices and mounted on silanized slides. PCNA (anti-PCNA; FL-261; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) was detected using a rabbit polyclonal antibody against amino acids 1-261 of human PCNA. For each slide, five images of the villus base were captured at 40× magnification. In these images, the number of PCNA-positive cells per square millimeter, as well as per crypt, were quantified.

Lesion Scoring in Intestine and Cecum

The severity of intestinal lesions was assessed at 21 (seven days post-infection) and 28 (14 days post-infection) days of age. Two birds per replicate, totaling 12 birds per treatment, were analyzed using a scale from 0 (absence of lesion) to 4 (severe lesion), following the criteria described by Johnson & Reid (1970) to identify the Eimeria spp. species involved.

Footpad pigmentation

Footpad pigmentation was evaluated using The Roche Colour Fan methodology, which allows quantification of pigmentation scores on a scale from 1 to 15. To standardize tegument coloration, all diets were supplemented with a commercial carotenoid at a concentration of 0.200 kg/ton. Evaluations were conducted at 19 and 28 days of age.

Statistical analysis

Data were subjected to analysis of variance (ANOVA) using the General Linear Model (GLM) procedure of the SAS statistical software (Statistical Analysis System). When significant differences were detected, means were compared using Tukey’s test (p<0.05).

RESULTS AND DISCUSSION

Growth Performance

Productive performance from 1 to 14 days of age is presented solely for the control programs, as the experimental challenge to which the birds were subjected occurred only at 14 days of age. The use of a nutritional additive in the diet and a coccidiosis vaccine had no significant effect (p>0.05) on average body weight, weight gain, feed intake, or feed conversion ratio during this period (Table 2).

Table 2
Productive performance of broilers, supplemented or not with nutritional additive, vaccinated against coccidiosis or not, before the enteric challenge.

1 to 14 days old
Programs	Live weight (g)	Weight gain (g)	Feed intake (g)	Feed conversion ratio
Control + AP	425.63	380.16	427.07	1.116
Additive + AP	421.78	376.67	427.70	1.125
Control + VA	424.04	376.23	432.09	1.123
Additive + VA	425.11	379.55	426.41	1.124
CV%	4.48	5.25	4.44	2.27
p-value	0.9614	0.9478	0.8826	0.8115

• AP: anticoccidial program; VA: anticoccidial vaccine

It is noteworthy that the inclusion of an early attenuated anticoccidial vaccine did not negatively affect the productive performance of broilers during the initial developmental phase. This was observed even though the birds had already undergone at least two endogenous cycles of the vaccine strain, involving both asexual and sexual replication of oocysts within enterocytes - an essential process for the development of effective cellular immunity.

According to McBride & Kelly (1990), maintaining the intestinal epithelium and associated supporting structures accounts for approximately 20% of the gross energy consumed by the animal. Therefore, it is plausible to infer that the use of a precocity-attenuated vaccine did not impair nutrient absorption through the intestinal mucosa. Cell renewal in the gut relies on nutrient availability, and a deficit in this process would likely result in diminished zootechnical performance, which was not observed.

As reported by Gadde et al. (2017), nutritional additives such as organic acids are considered excellent alternatives to antibiotic growth promoters due to their inherent antimicrobial properties. These compounds have demonstrated consistent benefits in both poultry and swine production. Accordingly, there is considerable expectation regarding their capacity to improve performance metrics (Chukwudi et al., 2025). However, it is important to acknowledge that in the present study, birds were reared under optimal sanitary conditions, received a balanced diet, and were maintained in a favorable environment. These factors may account for the absence of significant performance improvements resulting from dietary additive supplementation under such conditions.

An enteric challenge was administered at 14 days of age, and productive performance was evaluated during the subsequent period (14 to 19 days). No significant effects (p>0.05) were observed from the enteric control or challenge programs during this interval (Table 3). However, a significant impact of the challenge was detected between 19 and 21 days of age (p<0.05) (Table 3). Regardless of the program applied, challenged birds exhibited lower weight gain, lower feed intake (p<0.05) and higher feed conversion (p<0.07) compared to their unchallenged counterparts.

Table 3
Productive performance of broilers from 14 to 19 and 19 to 21 days of age, supplemented or not with a nutritional additive, vaccinated or not against coccidiosis, and submitted or not to enteric challenge.

Programs	Weight gain (g)	Feed intake (g)	Feed conversion ratio
14 to 19 days old
Control + AP	254.05	334.47	1.337
Additive + AP	264.25	351.72	1.271
Control + VA	278.60	359.02	1.314
Additive + VA	269.72	350.06	1.288
Enteric challenge
No challenge	270.36	353.47	1.303
With challenge	262.95	344.17	1.302
CV%	13.35	8.65	4.95
p-value
Programs	0.4027	0.2548	0.1795
Challenge	0.4754	0.2917	0.9452
Programs x challenge	0.1576	0.1674	0.4789
19 to 21 days old
Control + AP	244.26	312.96	1.319
Additive + AP	234.19	308.55	1.361
Control + VA	234.73	315.33	1.367
Additive + VA	228.81	310.10	1.332
Enteric challenge
No challenge	248.77a	322.86a	1.312b
With challenge	222.23b	300.62b	1.378a
CV%	11.21	5.17	8.88
p-value
Programs	0.5513	0.7646	0.7268
Challenge	0.0012	<.0001	0.0654
Programs x challenge	0.8691	0.1724	0.9201

• ^a-b
  Means in the same column followed by distinct lowercase letters differ from each other by Tukey’s test (p<0.05). AP: anticoccidial program, VA: anticoccidial vaccine.

In poultry, inflammatory responses can lead to intestinal tissue damage and consequently impair productivity (Broom & Kogut, 2018). Teng et al. (2020) evaluated the effects of a gradual Eimeria spp. challenge initiated at 13 days of age and observed that, between 13 and 19 days, challenged birds presented poorer feed conversion, as well as reduced weight gain and feed intake.

The present findings demonstrate that both the anticoccidial program and the early live vaccine provided comparable productive performance, regardless of the enteric challenge. This highlights the efficacy of the precocity-attenuated vaccine during the critical phase of immunity development.

There remains a widespread concern in the poultry industry regarding potential performance losses associated with vaccination against coccidiosis. According to Cervantes (2015), one of the primary apprehensions involves the possibility of attenuated vaccines causing considerable damage to the intestinal epithelium of broilers. Newman (1999) also reported that the use of attenuated vaccines has historically been associated with so-called “vaccine reactions,” characterized by intestinal lesions caused by parasite invasion and replication, ultimately leading to reduced zootechnical performance.

Nutritional additives are widely regarded as essential tools for enhancing performance in poultry production systems (Hankel et al., 2018).

For the cumulative period from 1 to 28 days of age, the enteric challenge significantly affected weight gain (p<0.05), with birds in the unchallenged control group exhibiting superior growth performance (Table 4). However, no significant effects (p>0.05) were observed for any of the coccidiosis control programs with respect to weight gain, feed intake, or feed conversion ratio. These results suggest that both the anticoccidial program and the early live vaccine were equally effective in controlling coccidiosis under the conditions of the present study.

Table 4
Productive performance of broilers supplemented or not with a nutritional additive, vaccinated or not against coccidiosis, and submitted or not to enteric challenge (1 to 28 days of age).

Programs	Weight gain (g)	Feed intake (g)	Feed conversion ratio
Control + AP	1479.98	1962.94	1.326
Additive + AP	1496.89	1954.95	1.307
Control + VA	1483.89	2001.33	1.348
Additive + VA	1505.19	1989.60	1.321
Enteric challenge
No challenge	1511.20a	2006.74a	1.328
With challenge	1471.77b	1947.67b	1.324
CV%	4.53	4.80	3.00
Programs	0.7846	0.5957	0.1004
Challenge	0.0500	0.0372	0.7268
Programs x challenge	0.1930	0.7156	0.3460

• ^a-b
  Means in the same column followed by distinct lowercase letters differ from each other by Tukey’s test (p<0.05). AP: anticoccidial program, VA: anticoccidial vaccine.

Ronsmans et al. (2015) demonstrated that broiler flocks managed with anticoccidial drug programs exhibited no significant differences in performance compared to those vaccinated with early attenuated vaccines. This aligns with the expected outcome of coccidiosis vaccination: with successive cycles of vaccine oocyst replication and shedding, field-resistant oocysts are gradually replaced by vaccine-derived oocysts that remain sensitive to anticoccidial drugs, thereby restoring drug efficacy over time.

Similarly, in a study by Mathis and Lang (2001), broilers vaccinated against coccidiosis-when not subjected to additional enteric challenges-showed compensatory weight gain by slaughter age (approximately 42 days), with final body weights equal to or greater to those of birds treated with anticoccidial drugs. This suggests that extending the evaluation period to 42 days, which corresponds to the typical commercial slaughter age in industrial poultry systems, might have revealed compensatory gains that were not observable within the 28-day window of the current study.

Another important consideration is that the use of anticoccidial vaccines eliminates concerns about chemical residues in poultry meat, which is a growing requirement from both consumers and regulatory bodies. This is one of the main drivers behind the search for effective alternatives to anticoccidial drugs in poultry production.

According to the most recent report from Brazil’s National Program for the Control of Residues and Contaminants (PNCRC; Brazil, 2024), monitoring results have consistently identified non-conformities in chicken meat samples related to the presence of nicarbazin, with levels exceeding the established Maximum Residue Limits (MRLs). These findings, recorded annually by the Ministry of Agriculture, Livestock and Supply (MAPA), further underscore the relevance of vaccination as a viable strategy to minimize the risk of residue violations and enhance food safety.

Intestinal Morphology

The results obtained for jejunal intestinal mucosa morphometry revealed a significant effect (p<0.05) at 19 days of age for villus width and absorptive area. Birds vaccinated against coccidiosis exhibited greater villus width (p<0.05) than those receiving the anticoccidial program, with or without the nutritional additive. However, for the absorptive area, birds that received the anticoccidial program showed superior values compared to vaccinated groups, regardless of additive inclusion (Table 5). Regarding the enteric challenge, challenged birds displayed significantly increased crypt width and a thinner muscular layer (p<0.05). Additionally, there was a significant interaction between coccidiosis control program and enteric challenge for crypt depth and villus:crypt ratio (Tables 6 and 7). These findings are consistent with those of Teng et al. (2020), who reported a 20% reduction in villus height in the duodenum and jejunum following an Eimeria spp. challenge.

Table 5
Morphometry of the jejunal mucosa of broilers, supplemented or not with a nutritional additive, vaccinated or not against coccidiosis, and subjected or not to an enteric challenge.

Programs	VH µm	VW µm	CD µm	CW µm	ML µm	VCR µm	ASA μm2
19 days
Control + AP	349.72	68.05b	40.62	29.22	60.65	8.73	10.97a
Additive + AP	348.21	69.43b	40.58	28.19	57.35	8.63	10.37ab
Control + VA	330.00	80.88a	42.65	29.37	51.72	8.09	9.35b
Additive + VA	331.25	75.09ab	42.78	29.80	55.54	7.94	9.21b
Enteric challenge
No challenge	337.01	72.29	39.48	28.32b	63.13a	8.90	10.05
With challenge	342.58	74.44	43.83	29.97a	49.50b	7.80	9.90
CV%	17.23	19.28	16.83	11.33	21.81	17.41	16.86
Programs	0.5255	0.0178	0.5977	0.4330	0.1277	0.2274	0.0023
Challenge	0.6513	0.4928	0.0059	0.0250	<.0001	0.0009	0.6789
Programs x challenge	0.7044	0.5419	0.0273	0.1038	0.4054	0.0307	0.4929
28 days
Control + AP	404.60bc	77.35	34.99b	31.75	64.37	11.10	11.74
Additive + AP	395.90c	75.50	36.97ab	29.34	66.86	11.28	10.91
Control + VA	483.58a	85.15	42.73a	31.78	72.33	12.77	11.22
Additive + VA	463.75ab	78.46	42.06a	31.22	71.71	12.20	10.74
Enteric challenge
No challenge	422.58	76.06	35.62b	30.59	66.48	11.94	11.89a
With challenge	451.33	82.16	42.76a	31.46	71.15	11.73	10.41b
CV%	19.74	25.44	17.75	11.41	18.50	24.21	18.72
Programs	0.0009	0.3773	0.0003	0.0634	0.1006	0.1520	0.3781
Challenge	0.1077	0.1476	<.0001	0.2336	0.0773	0.7290	0.0009
Programs x challenge	0.8508	0.5546	0.8184	0.4665	0.0761	0.8381	0.5381

• ^a-c
  Means within the same column followed by different letters significantly according to Tukey’s test (p<0.05). AP: anticoccidial program; VA: anticoccidial vaccine; VH: villus height; VW: villus width; CD: crypt depth; CW: crypt width; ML: muscle layer; VCR: villus:crypt ratio; ASA: absorptive surface area.

The interaction effects are further detailed in Table 6. In the unchallenged (control) groups, no significant differences were observed across treatments. However, in the challenged groups, crypt depth was significantly greater (p<0.05) in birds receiving the vaccine-based program compared to those on the anticoccidial program supplemented with an additive. Interestingly, the combination of vaccine and additive did not differ from other treatments, suggesting that the nutritional additive may serve as a beneficial adjuvant under challenge conditions by modulating the intestinal response.

Table 6
Breakdown of the interaction of the depth of the crypt of the jejunum (19 days), supplemented or not with nutritional additive, vaccinated against coccidiosis or not, and submitted or not to enteric challenge.

Programs
Enteric challenge	Control + AP	Additive + AP	Control + VA	Additive + VA	p-value
No challenge	38.90	41.13	36.69b	41.20	0.4706
With challenge	42.35AB	40.02B	48.60Aa	44.36AB	0.0328
p-value	0.2489	0.6065	0.0011	0.4336

• ^A-B
  Means followed by distinct uppercase letters on the line differ from each other. ^a-bMeans followed by different lowercase letters in the column differ from each other by Tukey’s test (p<0.05). AP: anticoccidial program, VA: anticoccidial vaccine.

Table 7 presents the interaction results for the villus:crypt ratio in the jejunum segment. Among unchallenged birds, no significant differences were found between treatments. In the challenged group, however, birds receiving the anticoccidial program, with or without the additive, showed a higher villus:crypt ratio than those vaccinated, either with or without the additive. This indicates that enteric challenge reduced the villus:crypt ratio in vaccinated birds, possibly reflecting heightened intestinal turnover and regeneration processes.

Table 7
Breakdown of the vilo:crypt ratio interaction of the jejunum (19 days), supplemented or not with nutritional additive, vaccinated against coccidiosis or not, and submitted or not to enteric challenge.

Programs
Enteric challenge	Control + AP	Additive + AP	Control + VA	Additive + VA	p-value
No challenge	9.01	8.62	9.34a	8.64a	0.6848
With challenge	8.45A	8.65A	6.85Bb	7.25ABb	0.0064
p-value	0.2707	0.9559	0.0018	0.0491

• ^A-B
  Means followed by distinct uppercase letters on the line differ from each other. ^a-bMeans followed by different lowercase letters in the column differ from each other by Tukey’s test (p<0.05). AP: anticoccidial program, VA: anticoccidial vaccine.

At 28 days of age, birds vaccinated against coccidiosis, with or without the nutritional additive, presented greater villus height and crypt depth (p<0.05) compared to birds on the anticoccidial program alone. This suggests that two weeks after the enteric challenge, the vaccinated groups-especially those also receiving the additive-exhibited a more robust regenerative response. The observed deepening of intestinal crypts, the primary site of enterocyte proliferation and differentiation, resulted in longer villi, a hallmark of improved nutrient absorption capacity.

From a zootechnical perspective, the results shown in Tables 2 and 3 indicate that vaccination is a viable strategy for coccidiosis control, with performance outcomes comparable to those of traditional anticoccidial drug programs. Importantly, birds vaccinated with early attenuated Eimeria strains had completed at least two endogenous replication cycles in the intestinal mucosa by 19 days of age. This did not compromise intestinal integrity; on the contrary, it appeared to stimulate early cellular immune responses without impairing the absorptive function of the intestinal epithelium or the overall productive performance of the birds.

Assessment of Intestinal Permeability via Serum FITC-d Levels and Immunohistochemical Assessment of Jejunal Cell Proliferation Using PCNA Marker

Gastrointestinal permeability was assessed by measuring serum levels of fluorescein isothiocyanate-dextran (FITC-d) recovered in the blood of broilers. At 19 days of age, a significant interaction was observed between the control programs and sanitary condition (Table 8). Upon analysis of this interaction, it was found that birds receiving the anticoccidial vaccine without nutritional additive supplementation exhibited lower FITC-d serum levels when subjected to the enteric challenge. However, when the vaccine was combined with the nutritional additive, no significant difference (p>0.05) in FITC-d concentration was observed between challenged and unchallenged birds (Table 9). The count of proliferating cell nuclear antigen (PCNA)-positive cells in the crypts of the jejunal mucosa was not significantly affected by either the control programs or the sanitary condition (Table 8).

Table 8
Serum FITC-d levels and cell count in mitosis of the jejunum of broilers at 19 days, supplemented or not with nutritional additive, vaccinated against coccidiosis or not, and submitted or not to enteric challenge.

Programs	FITC-d (μg/mL)	Cells in mitosis/mm2
Control + AP	0.21	0.625
Additive + AP	0.25	0.623
Control + VA	0.24	0.687
Additive + VA	0.23	0.732
Enteric challenge
No challenge	0.19	0.667
With challenge	0.27	0.670
CV%	27.49	30.17
p-value
Programs	0.4396	0.2549
Challenge	<.0001	0.9416
Programs x challenge	0.0387	0.8711

• AP: anticoccidial program, VA: anticoccidial vaccine.

Table 9
Unfolding of FITC-d interaction for broiler chickens, supplemented or not with additive, supplemented or not with nutritional additive, vaccinated against coccidiosis or not, and subjected or not to enteric challenge.

Programs
Enteric challenge	Control + AP	Additive + AP	Control + VA	Additive + VA	p-value
No challenge	0.22	0.28	0.33a	0.27	0.0816
With challenge	0.20	0.22	0.15b	0.18	0.3427
p-value	0.4223	0.1302	0.0009	0.0564

• ^a-b
  Means followed by different lowercase letters in the column differ from each other by Tukey’s test (p<0.05). AP: anticoccidial program, VA: anticoccidial vaccine.

FITC-dextran (FITC-d) is commonly employed in studies evaluating intestinal mucosal permeability, based on the principle that it is not absorbed from the intestinal lumen into the bloodstream under normal gastrointestinal tract (GIT) conditions. When the epithelium is compromised by damaging agents, lesions may disrupt cellular tight junctions (Awad et al., 2017), leading to increased intestinal permeability. This breakdown in barrier integrity can allow microorganisms or their components to translocate into the bloodstream, increasing the risk of systemic infections by opportunistic pathogens (Eichner et al., 2017).

In the present study, however, an inverse pattern of FITC-d translocation was observed. This unexpected result may be related to the life cycle of Eimeria spp. (Teng et al., 2018). These protozoa infect enterocytes and undergo development within these host cells (McDougald, 1998). According to Conway & McKenzie (2007), a single sporozoite can develop into a second-generation schizont capable of producing over 2.520.000 merozoites. This intracellular development causes enterocyte hypertrophy, which may reduce FITC-d permeability by increasing cell volume and potentially tightening intercellular space. Additionally, FITC-d sampling was conducted five days after the challenge (at 19 days of age), a period during which mucosal regeneration may have been initiated. Therefore, to better assess the relationship between intestinal permeability and enteric challenge with coccidial vaccine, FITC-d measurements taken during the later stages of recovery would be more informative.

PCNA is expressed during the late G1 and S phases of the cell cycle, and its detection using monoclonal antibodies provides a reliable method for evaluating cell proliferation without the need to administer exogenous markers (Rabenhorst et al., 1993).

Given that the nutritional additive used contained butyric acid-known to stimulate epithelial proliferation-a higher number of PCNA-positive cells was expected. However, this was not observed.

Rapid epithelial cell turnover is essential for maintaining and restoring the intestinal lining. Crypt depth is often interpreted as a compensatory response or epithelial hyperplasia, usually triggered by higher levels of mucosal damage due to enteric insults (Uni et al., 1998).

Intestinal lesion score and Footpad pigmentation

Regarding the intestinal lesion score, a significant effect (p<0.05) was observed for Eimeria tenella at 19 days of age. Birds receiving the anticoccidial program without the inclusion of the nutritional additive exhibited higher lesion scores compared to those vaccinated without additive supplementation. No significant differences were found among the other treatments. With respect to the enteric challenge, challenged birds showed significantly higher (p<0.05) maximum E. tenella lesion scores compared to the non-challenged group, regardless of the control program used (Table 10). At 28 days of age, no intestinal lesions were detected, indicating that the birds had fully recovered from the enteric challenge within two weeks, irrespective of the control program adopted.

Table 10
Intestinal lesion score and foot color of broilers, supplemented or not with nutritional additive, vaccinated against coccidiosis or not, and submitted or not to enteric challenge.

Programs	E. acervulina		E.maxima		E.tenella		Feet color
	19 days	28 days	19 days	28 days	19 days	28 days	19 days	28 days
Control + AP	0	0	0.40	0	0.54a	0	3.95	4.37
Additive + AP	0	0	0.62	0	0.20ab	0	4.12	4.50
Control + VA	0	0	0.25	0	0.16b	0	4.04	4.13
Additive + VA	0	0	0.50	0	0.20ab	0	4.31	4.12
Enteric challenge
No challenge	0	0	0.29b	0	0.20	0	4.17	4.42a
With challenge	0	0	0.59a	0	0.35	0	4.31	4.14b
CV%	0	0	162.92	0	161.18	0	14.74	14.36
Programs	0	0	0.3475	0	0.0173	0	0.2156	0.1109
Challenge	0	0	0.0537	0	0.1186	0	0.2435	0.0320
Programs x challenge	0	0	0.4248	0	0.3982	0	0.0191	0.0815

• ^a-b
  Means in the same column, followed by different letters differ from each other by Tukey’s test (p<0.05). AP: anticoccidial program, VA: anticoccidial vaccine.

Regarding foot pad pigmentation, a significant interaction (p<0.05) was observed at 19 days between the control program and sanitary condition (Table 10). Upon interaction analysis (Table 11), the unchallenged group that received the vaccine plus the nutritional additive showed greater foot coloration compared to the anticoccidial program. Within treatments, birds receiving the vaccine and additive exhibited reduced pigmentation under challenge conditions, whereas the opposite effect was observed for birds under the anticoccidial program without the additive.At 28 days, challenged birds showed lower (p<0.05) foot coloration regardless of the control program in question.

Table 11
Breakdown of the interaction of the color of the feet of broilers at 19 days, supplemented or not with nutritional additive, vaccinated against coccidiosis or not and submitted or not to enteric challenge.

Programs
Enteric challenge	Control + AP	Additive + AP	Control + VA	Additive + VA	p-value
No challenge	3.66Bb	4.33AB	4.08AB	4.63Aa	0.0047
With challenge	4.18a	3.90	4.00	4.0b	0.8060
p-value	0.0351	0.2201	0.7193	0.0263

• ^A-B
  Means followed by distinct uppercase letters on the line differ from each other. ^a-bMeans followed by different lowercase letters in the column differ from each other by Tukey’s test (p<0.05). AP: anticoccidial program, VA: anticoccidial vaccine

The use of vaccines for the control of coccidiosis in broilers is still not widely adopted in Brazil, facing resistance due to unsatisfactory outcomes in earlier studies involving vaccines composed of wild (non-attenuated) strains. These vaccines proved to be inefficient and overly aggressive, causing severe mucosal lesions and consequent impairments in zootechnical performance (Fabri et al., 2019). However, advances in vaccine development have led to the introduction of formulations based on attenuated strains, produced using modern technologies aimed at minimizing intestinal damage.

This is corroborated by the results of the present study, which demonstrate that the use of an early attenuated vaccine was effective in preventing intestinal lesions. The absence of aggressive lesions, historically associated with non-attenuated vaccines, highlights the efficacy and safety of current vaccine formulations in maintaining intestinal health and integrity. These next-generation vaccines, composed of all major Eimeria species attenuated by early lifecycle interruption, can promote robust cellular immunity with minimal disruption to intestinal morphology, and without negatively affecting broiler performance.

The pigmentation process depends on the intestinal absorption of dietary pigments, such as carotenoids included in the diet. These pigments are absorbed in the ciliated epithelium of the midgut following enzymatic hydrolysis, which requires a healthy intestinal mucosa. Conditions like necrotic enteritis or coccidial lesions compromise this process and may reduce pigment absorption.

According to Pantoja & Gonzales (2020), pigmentation is cumulative. These authors reported higher skin pigmentation in vaccinated birds compared to those under anticoccidial programs at 35, 42, and 49 days of age. Given that vaccination was administered on day one and the slaughter age in the present study was 28 days, it is plausible that similar pigmentation patterns would have emerged had the study continued until 42 days.

Dietary additives can support intestinal integrity and enhance pigmentation in broilers. Pantoja et al. (2020) evaluated the efficacy of an early attenuated vaccine in combination with short- and medium-chain fatty acids in broilers subjected to a necrotic enteritis and E. maxima challenge model. Their findings revealed that this combination reduced intestinal lesions, supporting the beneficial role of additives in conjunction with vaccination in promoting gut health and pigment deposition.

CONCLUSION

The exposure of birds to the experimental challenge compromised their zootechnical performance, caused morphometric changes in the intestinal mucosa, foot pigmentation and lesion score regardless of the control program used as compared to the unchallenged control group.

Both the anticoccidial program and the use of early attenuated vaccine, when associated with a nutritional additive composed of short and medium chain fatty acids, showed similar efficacy in relation to productive performance and intestinal crypt depth, showing that both strategies were effective in intestinal protection and productivity support.

Thus, the control of coccidiosis in broilers by means of an early attenuated vaccine associated with nutritional additives emerges as a viable, safe, and effective alternative to the use of traditional anticoccidials. This strategy allows the induction of early immunity without compromising intestinal integrity, favoring the absorption of nutrients and ensuring productive performance compatible with conventional programs, even under challenging conditions.

ACKNOWLEDGEMENTS

The authors would like to express their gratitude to the Poultry Experimentation Laboratory and to the Federal University of Paraná - Palotina for supporting the development of this project.

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• FUNDING
  This research received no external funding.
• DATA AVAILABILITY STATEMENT
  All available data are incorporated into the manuscript.
• DISCLAIMER/PUBLISHER’S NOTE
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Edited by

• Section Editor:
  Ramon Malheiros

Data availability

All available data are incorporated into the manuscript.

Publication Dates

• Publication in this collection
  27 Oct 2025
• Date of issue
  2025

History

• Received
  26 Apr 2025
• Accepted
  25 Aug 2025