Coccidiosis is a worldwide intestinal disease of chickens caused by protozoan parasites belonging to the genus Eimeria (1). Seven Eimeria species have been well-known to be responsible for avian coccidiosis (2, 3), and recently, three cryptic species designated as Operational Taxonomic Units (OTUs) X, Y, and Z, have been suggested and assigned by Blake et al. (4). These species of Eimeria exhibit varying degree of pathogenicity, with E. necatrix standing out as the most virulent species, while E. tenella is more prevalent, both inflicting bloody lesions, high morbidity, and high mortality in naïve chickens (5); E. brunetti is highly pathogenic and associated with hemorrhagic coccidiosis (6); E. acervulina, E. maxima, E. mitis, and E. praecox are usually less pathogenic, incurring malabsorption and enteritis (6). Avian coccidiosis can cause substantial financial losses, with an estimated global cost to the poultry industry approximately £10.36 billion annually (1).

Currently, the primary methods of controlling coccidiosis encompass preventative chemotherapy using in-feed anticoccidial drugs and live vaccines. However, the emergence of drug resistance in various regions worldwide has led to a reduction in the efficacy of anticoccidial agents (7). Additionally, concerns regarding the efficacy of live vaccine have been on the rise (8). In China, three types of live anticoccidial vaccines are available for use (National Veterinary Drug Basic Database Online),¹ in which two of them are attenuated (namely a trivalent vaccine containing E. tenella, E. acervulina, and E. maxima and a tetravalent vaccine containing E. tenella, E. necatrix, E. acervulina, and E. maxima). Another one is non-attenuated, known as Coccivac™ containing E. maxima, E. mivati, E. acervulina, and E. tenella. To evaluate the efficiency of poultry management, including the selection of Eimeria type in vaccines, a comprehensive understanding of the epidemiology of Eimeria species is essential.

Previous reports have monitored the patterns of oocyst accumulation in litter following vaccination (8, 9). The results have revealed a notable increase, marked by a small peak in the reproduction of vaccine strains between 2 and 4 weeks after vaccination at 1 week, followed by another slightly higher peak occurred at 4–7 weeks, which represented a challenge from the local virulent population. Subsequently, oocyst production reduced due to flock immunity after approximately 7 or 8 weeks, with no detectable oocysts remaining in the litter using conventional techniques. Similarly, changes in Eimeria oocyst concentration and species composition in litter of broiler farms subjected to various cycles of anticoccidial drug or live Eimeria oocyst vaccine control have been reported by Jenkins et al. (9). The influence of anticoccidial methods on the presence and composition of Eimeria species in the litter has also been underscored (10). These studies suggest that understanding the species composition in litter during live vaccine control may serve as a means for assessing the efficacy of a particular control program. However, comprehensive data regarding the prevalence and risk factors associated with Eimeria infection in broiler chickens administrated with live anticoccidial vaccines in China have been lacking.

Traditional classification of Eimeria species depends on morphological characteristics, the affected region of the intestinal tract, and the pre-patent period of Eimeria after passage through experimentally infected chickens. However, these methods may not achieve a species-specific diagnosis due to overlap in these characteristics among certain species (11). Recently, polymerase chain reaction (PCR) was used to identify all seven avian Eimeria species. These techniques employ genetic markers in the internal transcribed spacer-1 (ITS-1) (12, 13), ITS-2 (14), and sequence characterized amplified region (SCAR) (12). In the present study, we applied quantitative real-time PCR (qPCR) to conduct a comprehensive survey aimed at assessing the distribution of Eimeria species in broiler flocks vaccinated with live vaccine across China. This survey was designed to determine the prevalence of Eimeria infection and the risk factors in flocks.

Coccidiosis is a disease of significant economic concern worldwide in the poultry industry. The epidemiological monitoring of Eimeria species present in broiler chickens which are vaccinated with live vaccines is vital for the selection of prevention and controlling strategies for coccidiosis. The investigation in our study included seven well-known Eimeria species, excluding the three cryptic species OTU_X, OTU_Y, and OTU_Z. This survey was conducted based on the fact that the main prevalence species in China are the seven known species (16–19). Additionally, there have been few studies reporting the presence of unidentifiable OTUs in chickens immunized with transgenic Eimeria in China (20). The overall prevalence of coccidiosis in China is 86.12% (546 of 634 flocks). A comparably high prevalence has been reported in provinces in China such as Anhui (87.75%) (18), Hubei (97.79%), and Henan (96.70%) (16) and other countries including Greece (85.7%) (21), northeastern Algeria (99.5%) (22), Colombia (96.3%) (23), and Australia (98%) (24). Conversely, the prevalence was notably lower in Serbia, north India, Korean, northeastern Brazil, and southwestern Nigeria with rates of 59, 28.5, 75, 59, and 41.3% (25–29), respectively. The variance in Eimeria species occurrence can be attributed to differences in control methods, sampling times, animal management practices, and climatic conditions (30). Previous monitoring trials found that flocks fed with anticoccidial agent in diet produced a considerably higher count of the oocysts in vaccinated flocks, especially between 4 and 8 weeks (8). Consistently, our study also found that the prevalence in vaccinated flocks (86.12%) was lower than in flocks administered with anticoccidial agent (97.17%), as previously reported (16). Southern China’s warm and humid autumn climates, with an average temperature of 25°C and a relative humidity of 79%, may contribute to the elevated prevalence of Eimeria in broiler flocks. However, it is noteworthy that we did not find an increased number of Eimeria-positive flocks in southern China compared with the other three regions in China, suggesting more effective control measures being implemented in those areas.

Seven species of Eimeria were populated in broiler chicken farms across China. The most prevalent species included E. acervulina (65.62%), E. necatrix (50.95%), E. mitis (50.79%), and E. tenella (48.42%). Interactions among Eimeria species, coupled with crowding effect, are known as the most important factors affecting oocyst production (31). Notably, E. acervulina and E. tenella exhibit a higher reproductive potential, and in co-infections, E. acervulina reduces the oocyst production of E. necatrix, E. maxima, and E. brunetti (31). The mixed infection of E. acervulina–E. tenella (38.96%) and E. acervulina–E. necatrix (37.22%) was the dominant co-infection in our study. Interestingly, younger flocks vaccinated with tetravalent vaccines and aged younger than 4 weeks showed a higher likelihood of detecting positive for E. acervulina–E. tenella (50.47%, p < 0.05), E. acervulina–E. necatrix (52.34%, p < 0.05), and E. acervulina–E. tenella–E. necatrix (41.12%, p < 0.05) compared with older ones. This is in agreement with previous studies indicating that found younger chickens are more susceptible to Eimeria species (8, 30, 32). An exception was observed with E. maxima, in which the positive rate was extremely low (4.42%). This might be attributed to all the studied flocks being vaccinated with E. tenella, E. acervulina, and E. maxima or E. tenella, E. necatrix, E. acervulina, and E. maxima. One possible explanation for this could be that the primers for E. maxima failed to detect all strains present in the field samples, as demonstrated by Lew et al. (33) and Sun et al. (34). Alternatively, another reason may be some associated factors affecting the reproductive potential of the live E. maxima vaccine strains, since the positive rate of E. maxima was not higher in starter chickens compared with grower and adult chickens, especially in trivalent vaccines used flocks. This is inconsistent with the finding reported by Snyder et al. (35) that vaccinated flocks had peak oocyst shedding during weeks 2 to 4. Since the intestinal lesions caused by coccidiosis (especially E. maxima) were a well-known predisposing factor for necrotic enteritis (NE). The prevention of coccidiosis and NE was intimately connected due to the risk of NE was greater if the number of oocysts is excessive for E. maxima. The low frequency of E. maxima in this study might reduce the occurrence of NE in live vaccines used flocks. Nevertheless, it is worth noting that the relatively high prevalence of E. brunetti peaked at 21.61% in broiler chickens compared with the previous finding (6.6%) in China (16). Given its increased prevalence, E. brunetti may need to be included in the vaccine in China, echoing similar recommendation made in Australia as previous report (36).

Different Eimeria species distribution, control strategies, and geographical features could affect the occurrence of avian coccidiosis. E. necatrix is known to have lower fecundity and to be a ‘poor competitor’ compared with other species (8). However, In the case of tetravalent vaccinated flocks, the prevalence of E. necatrix was the highest in starter flocks (66.36%) while decreased in grower flocks (46.94%), followed by a slight increase in adult flocks (58.33%). Furthermore, there was a significant positive association between E. necatrix infection rate and coccidiosis occurrence (OR = 1.64, 95% CI: 1.07–2.51; p < 0.05), hinting that flocks might challenge by wild-type strains. This association was consistent with the finding identifying E. necatrix as a contributing factor to coccidiosis occurrence (8, 35). This finding suggested that factors related to the host (e.g., underlying subclinical non-parasitic infections) and environmental factors (e.g., crowding, air quality, and stress) may negatively affect the health of vaccinated chickens, which increased the susceptibility of chickens to coccidiosis, similar to previous reports (21, 29, 36). Moreover, our study found that flocks infected with E. acervulina were also at a significantly higher risk of coccidiosis (OR = 1.59, 95% CI: 1.01–2.51; p < 0.05) when vaccinated with tetravalent vaccines. Known for its rapid reproduction and short life cycle, E. acervulina’s preponderance in broiler farms and its link to coccidiosis outbreaks resonates with previous study (7). However, flocks vaccinated with trivalent vaccines and infected with E. maxima showed a significantly elevated risk of coccidiosis (OR = 7.07, 95% CI: 1.35–36.95; p < 0.05). This may be due to the vaccine strain of E. maxima that induced incomplete cross-strain immune protection against other wild-type strains (37). Surprisingly, flocks positive for E. praecox in tetravalent vaccine-immunized farms were also at an increased risk of coccidiosis (OR = 1.59, 95% CI: 1.02–2.47; p < 0.05). Despite often being overlooked, in our study, the high prevalence of E. praecox (41.80%) was consistent with previous survey in Henan and Hubei provinces in China (33.33%), Brazil (25.1%), and Australia (34.4%) (16, 24, 28). The pathogenicity of E. praecox was observed by Williams et al. for the first time to cause decrease in weight gain and increase in feed conversion ratio (38). Therefore, the high frequencies and risk associated with E. necatrix, E. acervulina, and E. praecox in broilers emphasize the importance of incorporating them into a comprehensive broiler vaccine.

In summary, suboptimal post-immunization poultry practices, following the administration of live attenuated vaccines, precipitate either isolated or combined infections of E. acervulina, E. necatrix, E. tenella, E. mitis, and E. praecox, whether as isolated or combined infections, leading to a pronounced susceptibility to morbidity. Furthermore, trivalent or tetravalent attenuated live vaccines are available in China. It is worth noting that the presence of new dominated E. brunetti and E. praecox could be included in the widely used live vaccines. Further investigations are needed to evaluate the epidemiology of virulent species in clinical contexts and discern their associated morbidity implications. By formulating an evidence-based immunization strategy and enhancing post-immunization poultry practices, we can regulate the prevalence of clinical coccidian taxa and assiduously curtail their affiliated pathological ramifications. Notably, our study is the first to report the prevalence of Eimeria species in flocks vaccinated with live vaccines in China, based on molecular analysis.

The original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding authors.

The animal study was approved by the Animal Care and Use Committee of the Institute of Animal Health, Guangdong Academy of Agricultural Sciences. The study was conducted in accordance with the local legislation and institutional requirements.

SL: Conceptualization, Data curation, Investigation, Software, Writing – original draft. XL: Conceptualization, Data curation, Investigation, Writing – original draft. QZ: Conceptualization, Investigation, Methodology, Writing – original draft. ZW: Investigation, Methodology, Writing – original draft. ZY: Investigation, Methodology, Writing – original draft. DW: Investigation, Methodology, Writing – original draft. GS: Investigation, Methodology, Writing – original draft. JL: Data curation, Methodology, Writing – original draft. ML: Data curation, Methodology, Writing – original draft. JH: Data curation, Methodology, Writing – original draft. HC: Data curation, Methodology, Writing – original draft. YS: Data curation, Methodology, Writing – original draft. XC: Data curation, Methodology, Writing – original draft. YZ: Data curation, Methodology, Writing – original draft. LY: Data curation, Methodology, Writing – original draft. JZ: Methodology, Resources, Supervision, Writing – original draft. NQ: Funding acquisition, Methodology, Project administration, Supervision, Writing – review & editing. MS: Funding acquisition, Methodology, Project administration, Supervision, Writing – review & editing.