4. Discussion
The purpose of the study was to investigate the effects of different
Eimeria inoculation doses on growth performance, litter moisture content, nutrient digestion and absorption, incidence and severity of FPD, gut microbiota, oocyst shedding,
Eimeria gene expression, and body composition in broilers raised in floor pens for 35 days. Cross contamination among pens in the
Eimeria infection studies in floor pens would be problematic [
27]. To minimize cross contamination between the unchallenged group and
Eimeria challenged groups, an anti-coccidial drug (Coban 90) was supplemented in the unchallenged group (CON group) in the current study. The dose of Coban 90 (500 mg/kg) was determined based on our previous study (unpublished). Broilers challenged with
Eimeria spp. and fed 500 mg/kg of Coban 90 showed improved body weight compared to the challenged group without Coban 90, and their BW was similar to that of the non-challenged group. Furthermore, the supplementation of 500 mg/kg of Coban 90 did not adversely affect the growth performance of broiler chickens without an
Eimeria challenge. However, although workers were extra careful during the entire experimental period, there was still
Eimeria cross contamination (e.g., oocyst shedding and lesion score) to the unchallenged group in the current study. These results indicate that supplementation of Coban 90 at 500 mg/kg did not completely inhibit the colonization of
Eimeria spp. in broilers even with indirect infection [
28]. However, significant statistical differences in body weight (BW) were observed between the control group and the
Eimeria challenged groups on Day 35. Additionally, throughout the current study, the control group consistently had lesion scores for
Eimeria spp. lower than 0.5 and a minimum gut permeability. These findings suggest that the level of Eimeria infection in the CON group did not have a significant impact on serving as the negative control for the CON group. The DFI was measured during the entire experiment period to check the severity of
Eimeria infection and whether
Eimeria re-infection occurred in floor pen conditions. The DFI could be one of the powerful and non-invasive parameters to indicate incidence and severity of
Eimeria infection in broilers. Measuring DFI has benefits in easiness and time over other non-invasive methods including measuring core body temperature or fecal moisture content. Our previous study [
29] showed that
Eimeria spp. Infection reduced ADFI in broilers in the acute phase (0 to 6 days post infection (dpi)). The time points 5 dpi and 6 dpi were considered as the peak time points for
Eimeria infection according to our previous studies based on the results of gut permeability and daily feed intake [
14,
29]. In the current study, DFI was dramatically decreased on 5 and 6 dpi and continued to be linearly reduced by increased
Eimeria inoculation doses from 11 to 14 dpi (D 26 to 29), which potentially indicates that re-infection of
Eimeria has occurred in the current study. This was supported by the results of
Eimeria lesion and oocyst shedding of
Eimeria spp. on D 28 and 35, while the average values for
Eimeria lesion scores on D 35 were below 1 out of 4 in the current study. Once exposed to
Eimeria infection, chickens develop strong humoral and cellular immunity against re-infection of
Eimeria [
30], which may demonstrate no statistical differences in gut permeability and intestinal morphology in broilers after the acute phase in the current study. Still, our current study showed that reinfection of
Eimeria was able to decrease feed intake of broilers in the floor pen conditions. Reduced feed intake in broilers infected with
Eimeria spp. might be due to alternation in immune response and endocrine system in broilers [
31,
32].
In the acute phase (0 to 6 dpi; D 15 to 21), reduced feed intake with impaired feed efficiency dramatically decreased BW and ADG in broilers infected with
Eimeria spp. in the current study, which was in agreement with Teng et al. [
14]. On D 21 to 28, BW and ADG were reduced, along with decreased ADFI, without affecting feed efficiency in
Eimeria-infected broilers. These results indicate that severe
Eimeria infection reduced growth rate of broilers by damaging the capacity of nutrient digestion and absorption and reducing the feed intake in broilers, but re-infection of
Eimeria decreased growth rate via decreasing only feed intake in broilers. These indicate that decreasing feed intake could be a sensitive sign in
Eimeria-infected broilers. On D 35,
Eimeria infection reduced only BW without affecting ADG, ADFI, and FCR in the
Eimeria challenged groups compared to the CON group on D 35. These results were in consistent with a study reported that
Eimeria inoculation on D 15 decreased BW in broilers on D 42 in floor pens [
33]. In previous studies, compensatory growth happened in broilers infected with
E. maxima in the recovery phase (6 to 13 dpi) [
12] and in broilers challenged with
E. acervulina and
E. maxima (14 to 21 dpi) [
34] by improving feed intake or feed efficiency after the acute phase of
Eimeria infection. However, potentially, infection of
E. acervulina,
E. maxima, and
E. tenella would not induce compensatory growth in the current study because they induced severe damage in the gastrointestinal tract of broilers. More studies are required to explain the potential mode of actions of
Eimeria infection on reduced feed intake and to elucidate compensatory growth after
Eimeria infection in broilers.
Higher
Eimeria inoculation doses above the threshold where the maximal reproductive potential reached may result in reduced oocyst shedding and downregulated
Eimeria genes relating to viability and sexual reproduction due to crowding effects in the gastrointestinal tract [
35]. In our previous study, while higher doses of
E. tenella increased oocyst shedding 5 to 6 dpi, oocyst shedding 6 to 8 dpi was not affected by challenging doses [
36]. This potentially designates that
Eimeria can control themselves to determine maximal oocyst production in broilers, which is called crowding effect [
35,
37]. In the current study, the ED4 group (the highest dose group) had numerically similar oocyst shedding of E. maxima and
E. tenella compared to the ED2 and ED3 groups in the cloaca content on D 22.
Eimeria oocysts were counted in the cloaca content and litter instead of feces because it is not feasible to collect fresh fecal samples in the litter condition. To elucidate crowding effects of
Eimeria spp., whole duodenal, jejunal, and cecal tissue samples were collected on 6 dpi, one day prior to peak date (7 dpi) for oocyst production of
E. maxima and
E. tenella [
38], and gene expression of
APN,
EF2,
GAM56, and
GAM82 of
E. maxima were modulated by increased
Eimeria inoculation doses along with quadratically modulated oocyst production of
E. maxima in the current study. The
APN,
EF, and
GAM of
Eimeria spp. play important roles in
Eimeria viability and sexual reproduction to produce oocysts [
24,
39,
40]. The inconsistency between linearly increased gene expression of
APN,
EF, and
GAM and quadratically modulated oocyst shedding of
E. maxima is still in question because only transcriptional level was analyzed in the current study. However, our current study showed that different inoculation doses of
Eimeria can alter
Eimeria gene expression. Increased inoculation doses only affected
E. maxima mRNA expression in the current study potentially because
E. maxima is more sensitive to crowding effects due to their largest size among chicken
Eimeria spp. [
41].
In the current study, ileal digesta and litter moisture contents were increased due to
Eimeria infection in the acute phase. In our previous study,
E. tenella infection did not increase ileal digesta moisture content on 5 to 7 dpi and even decreased ileal moisture content on 6 dpi [
36]. Increased ileal moisture content is mainly due to infection of
E. acervulina and
E. maxima in broilers. Increased digesta moisture content may imply shortened digesta transit time, which can reduce the capacity of nutrient digestion and absorption [
42] and is associated with reduced nutrient digestibility in the current study. Litter moisture content was linearly increased in the acute phase and was quadratically increased by
Eimeria infection on D 35 in the current study. Many studies demonstrated that litter moisture is closely associated with FPD in broiler chickens [
6,
43,
44]. FPD is a type of skin inflammation that induces necrotic lesions on the plantar surface of foot pad in broilers [
45]. FPD can reduce the marketability of chicken feet, be an entry route for pathogenic bacteria, cause lameness, and reduce the growth performance of broiler chickens [
46]. Increased litter moisture content due to
Eimeria infection can increase the incidence and severity of FPD in broilers by increasing the litter ammonia concentration [
47,
48]. The litter moisture content on D 35 and severity/incidence of FPD on D 35 showed similar trends among the experimental groups in the current study, while the severity and incidence of FPD was mild potentially because of the dry conditions of the room and early slaughter age (D 35) in the current study. A previous study by El-Wahab et al. [
43] showed that litter moisture should be above 35% to induce FPD in poultry.
Eimeria infection still has the potential to increase the incidence and severity of FPD by increasing litter moisture content in broilers.
Gut permeability measured by FITC-D4 is an important indicator to represent functionality and integrity of gut in broilers [
49]. Increased gut permeability indicates that more pathogens and toxins can permeate into the blood stream across the epithelial layer, which can cause systemic infection in broilers [
50]. In the current study,
Eimeria infection increased gut permeability on 5 dpi in broilers, which is consistent with our previous study [
14]. Our previous study demonstrated that
E. maxima infection had a significant impact on increasing gut permeability [
51], whereas
E. tenella infection did not affect gut permeability in broilers [
36]. Potentially, the infection of
E. acervulina and
E. maxima disrupts tight junction proteins and the mucus layer, which play an important role in maintaining gut barrier integrity, and this would make the intestinal wall thinner (more permeable) in broilers [
52,
53]. In contrast, Vicuña et al. [
49] showed that FITC-D4 can be deposited in the cecal tissue, and
E. tenella infection did not increase gut permeability potentially because
E. tenella infection thickened the intestinal wall in the ceca [
36]. No differences were observed in the gut permeability on D 27 in the current study, and this suggests that the re-infection of
Eimeria did not severely damage gut functionality and integrity, while it reduced feed intake in broilers in the floor pen conditions.
Reduced growth performance and feed efficiency might be mainly attributed to the reduced capacity of nutrient digestion and absorption in
Eimeria-infected broilers. In the current study, the AID of CP was linearly reduced by
Eimeria infection in broilers in the acute phase (0 to 6 dpi), which is consistent with our previous study by Teng et al. [
14], which reported that
Eimeria infection reduced the AID of CP in
Eimeria-infected broilers.
E. acervulina and
E. maxima are the main
Eimeria spp. that directly reduce the AID of CP because they inhabit in the duodenum and jejunum, respectively [
54,
55]. Our previous study by Choi et al. [
36] reported that
E. tenella infection in the ceca did not directly affect AID in the acute (6 dpi) and recovery phase (9 dpi) in broilers. The AID method does not account for the endogenous loss of nutrients in the gastrointestinal tract of broilers [
54]. Both nutrient disappearance and increased endogenous loss may have affected the AID of CP values in the
Eimeria-infected broilers because
Eimeria infection impaired the intestinal morphology and activities of jejunal brush border digestive enzymes in the current study.
Eimeria infection is known to increase endogenous losses of proteins (e.g., plasma proteins, mucin, and cell debris) in the gastrointestinal tract during
Eimeria colonization and reproduction activities [
56,
57]. The AID of CF was dramatically reduced by
Eimeria infection, and AID values of CF were negative in the ED2, ED3, and ED4 groups in the current study. Consistently, Ghareeb et al. [
58] reported that
E. maxima infection dramatically reduced the AID of CF in broilers. The negative values for AID of CF indicate that there were high endogenous losses in
Eimeria-infected broilers. Endogenous fat from the gastrointestinal tract includes bile, cell debris, intestinal secretions, and microbial lipids [
59]. Adams et al. [
60] demonstrated that infection of
E. acervulina decreased the secretion of bile, which has an essential role in fat digestion by emulsifying fat in the gastrointestinal tract. Potentially, reduced bile secretion and increased cell debris from the gastrointestinal tract may have decreased fat digestibility and increased endogenous loss of fat, which resulted in negative values for the AID of CP in
Eimeria-infected broilers in the current study. However, more studies are needed to specify the factors such as bile, cell debris, intestinal secretions, and microbial lipids that increased endogenous losses of fat in
Eimeria-infected broilers.
Intestinal morphology is an important indicator to represent capacity of nutrient digestion and absorption in the gastrointestinal tract of chickens [
61]. In the current study, infection of
E. acervulina and
E. maxima reduced VH and increased CD in both duodenum and jejunum on D 21. These results agree with several previous studies [
62,
63]. The reduced VH and increased CD implies there was high tissue turnover in the intestine by
Eimeria infection because bigger crypts, which are reservoirs for enterocytes, indicates a high demand for new tissue in the villus [
62,
64]. Impaired jejunal morphology is highly associated with reduced activities of jejunal maltase in the current study. This is because mature enterocytes in the villus, which mainly express brush border digestive enzymes, would be quickly deceased due to a higher turnover rate in
Eimeria infection conditions [
65]. Furthermore, negatively modulated duodenal and jejunal morphology would explain decreased AID of CP and EE in the current study. While no differences were observed in the jejunal morphology on D 35, duodenal VH:CD were quadratically reduced with increased duodenal CD in
Eimeria-infected broilers in the current study. Reduced VH:CD due to increased CD indicates more energy and nutrients are required for gut maintenance, which can result in growth retardation in broilers [
62]. However, while statistical differences were not observed, the ED3 and ED4 groups exhibited numerically higher duodenal VH and numerically lower VH:CD compared to the control group on D 35. This numerical trend suggests that gut maintenance and new tissue generation may have occurred on D 35. This result also indicates that negative effects of
E. acervulina infection lasted through to D 35 in broilers in the current study.
Chicken ceca have only crypts without villus, similarly to the colon of mammals (humans and pigs) [
66]. In the current study,
E. tenella infection deepened the cecal crypts as we observed in our previous study [
36]. However, it is still uncertain whether thickened cecal wall due to
E. tenella infection decreases the permeation of microbial metabolites (e.g., endotoxins and short chain fatty acids) across epithelium. In the current study,
Eimeria infection negatively affected cecal microbiota in the acute and chronic phases. In the current study,
Eimeria infection increased the phylum Proteobacteria and the family Enterobacteriaceae in the acute phase, which includes diverse pathogenic bacteria such as
Escherichia coli,
Salmonella spp.,
Vibrio spp., and
Pseudomonas spp. [
67]. Moreover, in the acute phase,
Eimeria infection reduced the relative abundance of the family Christensenellaceae and Peptostreptococcaceae, which play important roles in fiber degradation and short chain fatty acid production in broilers [
68]. Potentially, negatively altered cecal microbiota may have reduced volatile fatty acids (VFA) production in the ceca, and this would explain the reduced activities of SAP, which needs VFA production [
36,
69]. Whereas alpha diversity indices in the cecal microbial communities on D 21 were not affected by
Eimeria infection, all alpha diversity indices including pielou evenness (evenness), faith phylogenetic diversity (biodiversity based on phylogeny), shannon entropy (richness and evenness), and observed features (richness) were linearly decreased on D 35 in the current study. Reduced alpha diversity indices suggest the presence of unhealthier and immature gut microbiota in chickens [
70]. Furthermore, the relative abundance of the family Enterobacteriaceae was quadratically increased by
Eimeria infection, and the relative abundance of the family Ruminococcaceae, which play an important role in fiber degradation and short-chain fatty acid production in the ceca of chickens [
71], was linearly reduced by
Eimeria infection on D 35 in the current study. These results imply that
Eimeria infection on D 15 still negatively influenced cecal microbiota in broilers on D 35. Our previous study showed that
E. tenella infection negatively influenced cecal microbiota toward increasing the abundance of pathogenic bacteria and reducing microbial VFA (e.g., an important energy source for chickens) production mainly by impairing the mucosal immune system of broilers and increasing the protein concentration in the cecal content [
72]. While
E. tenella infection would be the main factor to alter cecal microbiota, the infection of
E. acervulina and
E. maxima potentially can affect cecal microbiota by increasing endogenous loss of proteins and undigested dietary proteins in the gastrointestinal tract because the phylum Proteobacteria mainly ferment protein sources for their growth and reproduction [
73,
74,
75]. More studies are required to investigate whether the infection of
E. acervulina and
E. maxima itself can alter cecal microbiota in broilers.
Body composition is a crucial parameter in broiler production because body composition is closely associated with meat yield and quality [
76]. In the current study, body composition of broilers was altered by
Eimeria infection. On D 35, the lean:fat ratio was linearly enhanced, and fat accumulation was reduced by increased
Eimeria infection doses in the current study. Potentially,
Eimeria infection stimulated the immune system, resulting in the excessive usage of energy sources (e.g., fat and glycogen) in broilers [
77,
78]. Furthermore, reduced fat digestibility and decreased cecal VFA production due to negatively altered microbiota on D 21 could result in decreased fat accumulation as a chronic effect on D 35. These results suggest that
Eimeria infection can influence the body composition and meat quality of broilers.