One-day-old Ross 708 broiler chicks were obtained from a local hatchery (Sofina Foods) and housed in the Poultry Research Center at the University of Alberta, Edmonton, Canada. The natural NE infection model was developed stepwise using a total of 752 animals from three experimental flocks. Animals in flocks 1 and 3 were randomly assigned to two dietary treatments to evaluate the impact of antibiotic removal on NE development (flock 1: antibiotic treatment with 21 cages of 8 birds, and drug-free treatment with 22 cages of 8 birds; flock 3: antibiotic treatment with 8 pens of 18 birds, drug-free treatment with 8 pens of 18 birds). Animals in flock 2 were used for evaluating the immunomodulation effect of β-glucan and were randomly assigned to three injection treatments (each with 5 cages of 8 birds). Flocks 1 and 2 were reared in Specht pullet cages (21 × 23.5 × 17.5 inches, Specht Canada Inc.), and flock 3 was housed in the floor pens (0.9 m × 1.4 m). All three flocks were treated with a natural NE challenge procedure but with different levels of coccidiosis challenge intensity (Table 3).

Birds from all three flocks were fed a wheat-based diet formulated to meet or exceed the management guide recommendations for all nutrients. The experimental diets were administered as a starter diet, grower diet, and finisher diet (Table 1). The diet composition for flock 3 was adjusted as part of an adaptation of the model to include a more practical commercial-type diet. Feed and water were provided ad libitum. Temperature and lighting were monitored daily and adjusted according to the Ross 708 guidelines (Aviagen, 2019). On day 13, a 10× (flock 1) or 15× (flocks 2 and 3) dose of the Coccivac-B52 vaccine (Merck Animal Health) containing live, sporulated Eimeria oocysts (E. acervulina, E. mivati, E. tenella, and two strains of E. maxima at unspecified doses) was administered through oral gavage. Each bird received 1 ml of vaccine diluted in distilled water. On day 18, the feed was withdrawn for 24 h with animals being closely monitored for health over the subsequent 3 days. Figure 2B shows the predisposing factors application timeline in the natural NE challenge model.

For experimental flocks 1 and 2, animals were randomly selected and examined for NE disease status on days 17, 21, and 40 (flock 1: n = 16, flock 2: n = 18). Flock 3 was sampled on day 21 and day 40 (n = 128). The NE-specific lesions in the small intestine were scored as described by Shojadoost et al. (2012) with some modifications. Animals were scored from 0 to 3 based on the following criteria:

0: No gross lesion;

1: Thin or friable walls, or diffuse superficial fibrin;

2: Focal necrosis or ulceration, or non-removable fibrin deposit;

3: Multifocal necrosis or ulceration, or nonremovable fibrin deposit.

Lesions more severe than score 3 were not observed.

Following lesion scoring, a 3 cm intestinal section at the lesion site was collected and fixed in 10% formaldehyde for microscopic histology analysis. Fixed intestine tissue was dehydrated and embedded in paraffin wax, then sliced into 5 μm sections and stained with hematoxylin and eosin. The intestinal cross sections were examined under light microscopy. Images were collected by using a SeBaCam digital microscope camera with SeBaView software (Thermo Scientific).

Statistical analyses were performed by the GraphPad Prism 8 software. The experimental unit was the individual bird. To understand the impact of the coccidial challenge on natural NE development, Fisher’s exact test was conducted to compare the lesion score between low- and high-coccidial challenged animals. A nonparametric Mann-Whitney test was used to compare the rank of lesion score between two coccidial challenge levels of the same age. T-tests were conducted to compare bacteria abundance between the age of days 17 and 21. p < 0.05 was defined as being statistically significant.

The two cage-reared flocks yielded similar mortality during the experiment period. The overall mortality (day 1–40) was 1.15% in flock 1 and 1.39% in flock 2 (Table 3). All mortalities occurred within the first week, prior to the coccidial vaccine dosing and feed withdrawal challenge. Birds did not show observable clinical signs, but bloody and mucous-containing feces were found after feed withdrawal, indicating the presence of diarrhea.

The floor-reared flock (flock 3) showed higher overall mortality at 3.82% compared to the cage-reared flocks (Table 3). Two mortality peaks were observed during the experiment period. The first peak occurred during week 1 and the second peak was found between weeks 3–5 after the 24-h feed withdrawal was applied. The second mortality peak was directly triggered by feed withdrawal on day 18, which also caused depression and decreased mobility in birds.

Cecal total C. perfringens was quantified by qRT-PCR targeting the 16 s gene. In flock 1, all sampled birds were found to be C. perfringens positive regardless of age (Figure 3A). The presence of netB, the hallmark of NE-causing strains, was detected in 75.0% of the samples on day 17, before the feed withdrawal challenge, and increased to 93.8% on day 21. Correspondingly, netB abundance on day 21 was significantly higher than day 17 (p = 0.0242) (Figure 3B). This is consistent with our expectation that the 24-h feed withdrawal on day 18 further contributed to the propagation of the virulent strains within the flock. The observed C. perfringens density was relatively high with 16s abundance ranging from 10⁷ to 10⁹ copies per gram of cecal content (Figure 3B). A relatively lower netB abundance was observed at around 10⁶ copies per gram of digesta. Quantification of 16 s and netB copies showed no significant difference between day 17 and day 21 (p > 0.05), though day 21 tended to have a higher abundance of the examined genes. Flock 2 was reared in similar conditions as flock 1 but was challenged with a higher dosage of Eimeria oocysts on day 13. However, the higher coccidial challenge level did not increase bacteria detection rate or bacteria abundance (data not shown).

In our study, mild lesions (scores 1 and 2) were predominantly observed with a few birds scored with 3 (Figure 3E). Severe lesions (score greater than 3) was not observed in any of the flocks. A total of 48 birds were sampled in experiment flock 1. Only 5 birds (10.42%) had NE-specific lesions under the 10× coccidiosis vaccine challenge (Table 3), and all the lesion-positive animals were observed on day 21. With increased intensity of coccidial challenge at (15× vaccine dose), flock 2 (n = 54) and flock 3 (n = 256) showed a higher prevalence of birds with necrotic lesions (85.19 and 80.08%, respectively). Lesion prevalence in flocks reared in the wire-floored cage environment (flock 1 and 2) showed that coccidial challenge levels have a profound impact on NE lesion development (Figure 3C). The 15× dosage of Eimeria vaccine gavage led to prevalent lesion development in the flock as early as day 17 and the lesions were also present on day 40. The high-coccidial challenge flock showed increased lesion score compared to the low-challenge flock on day 17 (p < 0.0001), day 21 (p = 0.0045), and day 40 (p < 0.0001) (Figure 3D). The floor-rear flock (flock 3) with 15× coccidial challenge yielded consistently higher lesion prevalence on both sampling days as expected. The observed lesion prevalence was 75% (96/128) on day 21 and 93.75% (120/128) on day 40. Animals in flock 3 were not sampled on day 17.

Representative histopathological images of the intestinal section are shown in Figure 3E. Intestine tissue scored at 0 with no gross lesion and generally showed intact villus structure. However, examination under higher magnification revealed pathological changes including the presence of Eimeria oocysts, mildly dilated capillaries, and capillary hemorrhage. Tissue lesions that scored 1 and 2 generally showed similar microscopic appearance though distinguished changes were observed in gross examination. Under microscopic examination, the necrotic region showed hyperemia, villus fusion, and separation of epithelium from the lamina propria. The necrotic tissue was usually covered by adherent fibrin and cellular debris. These pathological alterations were also observed in lesioned tissue with a score of 3. Noticeably, sloughed mucosa leading to complete loss of villi was observed in certain areas within the lesioned tissue (Figure 3E).

Bacteria quantification together with the characteristic pathology of NE, such as clinical signs and gut lesions, is indicative of successful induction of NE disease (Williams et al., 2003; McReynolds et al., 2004; Palliyeguru et al., 2010). C. perfringens can be found in high populations in NE-affected animals ranging from 10⁶ ∼ 10⁹ CFU (Williams, 2005; McDevitt et al., 2006; Abildgaard et al., 2010). The C. perfringens population observed in our trial is consistent with those typically found in NE-affected animals. Interestingly, the netB gene was more prevalent on day 21 compared to day 17 (Figure 3A). This may be associated with the observed diarrhea on days 18–19 following the feed withdrawal challenge, which could indicate that the experimental conditions promoted the spreading of the netB-carrying strains in the flock.

As noted in previous studies, there may be a poor correlation between the number of C. perfringens organisms in the digesta and the incidence or severity of necrotic enteritis, especially in the subclinical form of the disease (Fernando et al., 2011). Subclinical NE is usually mild with no clinical signs or sudden increase in mortality (Fernando et al., 2011). Thus, gut lesions are considered to be a sensitive disease indicator compared to clinical signs and mortality (Williams et al., 2003). In this study, gut lesions were found in all three experimental flocks and were confirmed with microscopic examination (Figure 3E). Together, the observed bacterial load, clinical signs, and pathological changes suggest NE occurrence in the flocks with disease severity peaked during weeks 3 to 4.

Many conventional NE disease models have shown lesion incidence peaks at a certain age and declines as the animal approaches slaughter (Lovland et al., 2003; Gholamiandehkordi et al., 2007). Natural NE infection induced by reused litter material from a previous flock (Palliyeguru et al., 2010), high stocking density, and housing of birds on litter (Lovland et al., 2003; Fernando et al., 2011) have resulted in lesion prevalence ranging from 6.9 to 68.6%. However, to our knowledge, most studies conducted only one lesion examination during the rearing period. In the field conditions, subclinical NE-affected animals can be detected at slaughter with C. perfringens-associated lesions in the liver and gut (Lovland and Kaldhusdal, 1999; Johansson et al., 2010). This suggests experimental NE models with persisting lesion occurrence may better reflect NE cases in the field, where birds suffering from the subclinical disease are kept without being treated. In this study, we thus involved multiple gut examinations throughout the rearing period. Results suggested coccidial challenge has a profound influence on the development of gut NE lesions. Coccidial challenge intensity affects the occurrence and duration of gross lesions present in the NE-affected flocks. As noted by Stanley et al. (2014), Eimeria spp. caused significant changes in gut microbiota diversity and enabled C. perfringens to persist post challenge. C. perfringens inoculated in the absence of this predisposing factor fail to establish and maintain themselves in the gut flora.

Epidemiology studies suggest that NE occurrence is associated with specific housing conditions, including access to litter and floor-type housing (Kaldhusdal et al., 2016; Goossens et al., 2020). Our data shows NE-related mortality was not observed in our cage-reared flocks even with a 15× coccidial challenge dosage. A sudden but minor increase in mortality in the floor-reared flock was observed starting from day 18. In the E. maxima/C. perfringens dual infection model, NE lesions can be produced without mortality in animals reared in wire cages (Williams et al., 2003), while similar models in floor-reared broilers yielded NE-induced mortality ranging from 8 to 12% (Wu et al., 2010; Hofacre et al., 2019).

Natural NE infection induced by reused litter material from a previous flock resulted in mortality from days 15 to 30, ranging from 1.5 to 4.9% across dietary treatments (Palliyeguru et al., 2010). Fernando et al. (2011), by housing birds on wood-shaving litter and removing antibiotics, induced NE infection with mortality of 1.19–1.66% from days 20 to 36. These findings and our observation are consistent with the mortality range reported in subclinical NE affected flocks (Lovland and Kaldhusdal, 2001). Recent work by Calik et al. (2019) and Emami et al. (2019) described a new natural NE disease model by spraying the same coccidiosis vaccine, as used in our study, on litter and feed upon bird placement. An NE outbreak was observed on days 7–9 with overall mortality at around 12%. These findings showed higher mortality which peaked at an earlier age compared to our study but is consistent with our observation that the NE outbreak occurred 1–2 weeks after the coccidial challenge by concentrated Coccivac^®-B52 vaccine.

Conventional clinical NE models usually involve repeated oral inoculations of coccidial oocysts and C. perfringens for consecutive days (Williams et al., 2003; McReynolds et al., 2004; Gholamiandehkordi et al., 2007; Park et al., 2008; Wu et al., 2010; Jayaraman et al., 2013). One of the advantages of the natural NE model is the simplicity of the challenge schedule. Coccidial inoculation typically takes 30 min for two experienced technicians to gavage 100 birds, while feed withdrawal can be done within an hour even in large flocks. Experience from our research facility showed consistent induction of NE across studies using this protocol. Reduced complexity in the challenge schedule can limit animal stress and treatment inconsistency between different personnel, thus contributing to persistent induction of NE disease.

This natural infection model also allows flexibility in designing dietary formulas. The wheat-based broiler chicken diets commonly include xylanase to break down arabinoxylans, decrease viscosity, and increase digestibility in the birds (Lee et al., 2017). Elimination of xylanase has been used to produce natural NE infection (Abildgaard et al., 2010), by increasing feed transit time promoting C. perfringens persistence (Choct et al., 2006). Another commonly used dietary predisposing factor, fishmeal, supplies abundant glycine and methionine that enhance C. perfringens proliferation and toxin production (Wilkie et al., 2005; Dahiya et al., 2007; Shojadoost et al., 2012). However, the usage of fishmeal is limited in broiler feed due to its high cost and low availability (Frempong et al., 2019). We showed that birds fed with a fishmeal-free diet with xylanase inclusion (flock 3) showed prevalent NE lesions in the gut, suggesting fishmeal inclusion and xylanase elimination are not required for experimental NE induction.

The presence of pathogenic C. perfringens is required but insufficient to trigger NE infection. The virulence phenotype of C. perfringens is subject to the influence of host epithelium and complex lumen environment (Figure 1). This highlights the cooperative roles of a wide range of environmental factors that contribute to NE development. For research aimed at prophylaxis of NE, it is critical to conduct the evaluation under a condition accurately mimicking the disease development under practical production conditions. The infection model presented in this study was able to reproduce the commonly observed subclinical NE, regarding the severity of symptoms, timing of lesion development, and rate of mortality. By allowing the pathogen to develop in vivo, NE researchers will be able to evaluate a prophylactic strategy at an early stage of disease development, when the damage to the animal is most reversible and thus ideal to be targeted. However, more studies are needed to better understand this novel infection approach, such as alterations to mucus characterization, epithelial properties, and immunological function during natural NE induction.

Feed withdrawal introduced on day 18 in this natural NE challenge is important to trigger the timely development of the disease outbreak. At the same time, this approach is believed to cause limited stress to the animals and is considered humane when used properly. Pathogenic C. perfringens has a stronger ability in binding extracellular matrices and utilize nutrients released from host intestinal tissue, thus showing a selective survival advantage over nonpathogenic strains during feed withdrawal (Timbermont et al., 2009, 2014; Wade et al., 2015). Feed restriction is often used in conventional NE challenges, not as a designated stressor but as a measure to ensure uniformity of inoculation treatments (Shojadoost et al., 2012). In those situations, the C. perfringens inoculation is administered mixed with feed, and feed is usually withdrawn overnight so that the birds will eagerly eat the inoculated feed.

The NE-causing C. perfringens are of high diversity in terms of genomic content, with the varied ability to cause intestinal damage. The growing understanding of the differences between strains isolated from animals of different health statuses and geographical regions highlights the need to carefully select appropriate strains to use in experimental NE models. It was reported that 2 C. perfringens strains, both isolated from NE-affected chickens and carrying NetB, showed varied virulence and produced different levels of disease severity in experimentally-induced NE (Gharib-Naseri et al., 2019). Knowledge gained using this challenge model can likely be applied across wide geographic regions. The natural, subclinical infection NE challenge model would allow the propagation of pathogenic strains that are locally prevalent. This would be beneficial to the specific region in the understanding of pathogenesis and control strategies against the locally prevalent pathogenic strains.