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. 2023 Jun 5;102(8):102845. doi: 10.1016/j.psj.2023.102845

Pathogenicity and drug resistance of the Eimeria tenella isolate from Yiwu, Zhejiang province, eastern China

Hongchao Sun ⁎,1, Xinyao Su ⁎,†,1, Yuan Fu *, Lili Hao †,2, Wei Zhou ‡, Zhijin Zhou ‡, Jing huang ‡, Yimin Wang §, Tuanyuan Shi ⁎,2
PMCID: PMC10404733  PMID: 37441842

Abstract

Chicken coccidiosis can cause severe enteritis with high mortality, which causes serious economic losses to the global breeding industry each year. The most virulent species is Eimeria tenella (E. tenella), but the infectivity of different E. tenella varies among geographic strains. At present, there are no reports related to the pathogenicity and drug resistance of E. tenella in Yiwu, Zhejiang province, China. A total of 600 fecal samples were collected from 10 farms in Zhejiang province, the overall oocyst prevalence was 54.2% (325/600). The prevalence was significantly higher (P < 0.01) in chickens under 40 d (97.5%) than that in chickens between 60 and 85-days-old (40.5%) and chickens over 90-days-old (24.5%). E. tenella stain was isolated from fecal samples of chickens in Yiwu and the pathogenicity of this isolate was determined, and then we recorded the survival rate, bloody stool score, lesion score, average weight gain. The results showed that all of the chickens infected with 5 × 105 sporulated oocysts of E. tenella died after the seventh day of infection, the bloody stool score and average lesion score of chickens from group 1 (5 × 105), group 2 (5 × 104), group 3 (5 × 103) and group 4 (5 × 102) decreased successively; the average weight gain (g) and relative weight gain (%) increased successively; the weight gain of the low-dose E. tenella infection groups (5 × 103 and 5 × 102) were higher than the other 2 groups (5 × 105 and 5 × 104) (P < 0.05). Finally, The E. tenella isolate was tested for sensitivity to 6 anticoccidial drugs (sulfachloropyrazine sodium, amproline, toltrazuril, clopidol, salinomycin, and nicarbazine) using 4 indexes including anticoccidial index(ACI), percent of optimum anticoccidial activity (POAA), reduction of lesion scores (RLS), and relative oocyst production (ROP). The results showed that this isolate has developed severe resistance to drugs of salinomycin and nicarbazine, moderate resistance to amproline and clopidol, slight resistance to toltrazuril, while the E. tenella isolate performed more sensitive to sulfachloropyrazine sodium.

Key words: Eimeria tenella, isolate, pathogenicity, drug resistance

INTRODUCTION

Coccidia is an obligate intracellular protozoan parasite belonging to the genus Eimeria that causes coccidiosis, which has been recognized as a serious threat to the livestock industry worldwide (Matsubayashi et al., 2020) especially on the poultry industry (Burrell et al., 2020), leads to an estimated loss of more than 3 billion USD annually worldwide (Blake and Tomley, 2014). The clinical symptoms of coccidiosis include diarrhea, apathy, and inflammation of the infected intestine (Tomczuk et al., 2015), and even death in severe symptoms (Wang et al., 2019a). At present, 7 species of Eimeria have been reported in chickens (Cheng et al., 2022), they are named Eimera necatrix, Eimera tenella, Eimera brunetti, Eimera maxima, Eimera acervulina, Eimera mitis, and Eimera praecox, chickens of different ages and breeds are susceptible to coccidia. Among these, E. tenella is one of the most virulent species (Saidi et al., 2021), which specially targets the ceca of chickens and causes hemorrhagic pathologies (Okumura and Takeda, 2017). In broilers, even mild subclinical symptoms can reduce the feed efficiency (Arabkhazaeli et al., 2013).

Currently, anticoccidial drugs in the diet of poultry is one of the main methods to control Eimeria infection (Tan et al., 2017), although anticoccidial drugs are widely applicable against different Eimeria species (Wunderlich et al., 2014), problems such as drug resistance and drug residue in meat still can't be solved (Wang et al., 2019b). In European countries, since 2006, the use of anticoccidial drugs as feed additives has been strictly restricted (Yang et al., 2015). At present, ionophore and synthetic drugs are still the main choices for controlling coccidiosis (Min et al., 2004). Ionophore drugs mainly include salinomycin, monensin, and iamicin. Chemical synthetic drugs include a wide range, such as sulfachloropyrazine sodium, amproline, toltrazuril, clopidol, and nicarbazine (Greif et al., 2001; Chen et al., 2018). The anticoccidial drugs can destroy membrane integrity (Qaid et al., 2021), inhibit protein synthesis, inhibit DNA synthesis (Wunderlich et al., 2014) and induce swelling of mitochondria and endoplasmic reticulum (Darius et al., 2004).

Owing to the long-term use of anticoccidial drugs, significant drug resistance of Eimeria spp. strains has developed gradually (Blake and Tomley, 2014), drug resistance could limit the effectiveness of these kinds of drugs. In this study, an epidemiological investigation of Eimeria was performed in chickens in Zhejiang province, China. Next, an E. tenella Yiwu strain was isolated and the pathogenicity of this isolate was studied. Finally, drug resistance was identified using 6 anticoccidial drugs. Our results can be used to develop guidelines for using of anticoccidial drugs properly.

MATERIAL AND METHODS

Ethics

All protocols were approved by the Zhejiang Academy of Agricultural Sciences Institutional Animal Care Committee in accordance with the recommendations of the National Institutes of Health Guide for the Care and Use of Laboratory Animals (Ethics protocol No. 2022ZAASLA67).

Animals and Drugs

The 1-day-old specific pathogen free (SPF) chickens using in this experiment were purchased from a local poultry-breeding center (Xiaoshan, Hangzhou, China). These chickens were raised in an insulation house (30°C–37°C) and offered forage (without any anticoccidial drugs) and cold boiled water, the forage was baked in a 70°C oven for at least 2 h before feeding to the chickens.

The anticoccidial drugs (sulfachloropyrazine sodium, amproline, toltrazuril, clopidol, salinomycin, and nicarbazine) were purchased from Guangdong Wens Dahuanong Biotechnology Co, Ltd (Yun Fu City, Guangdong province, China).

Samples collection and Prevalence Survey of Coccidia

A total of 500 fresh fecal samples were collected from Lishui, Taizhou, Hangzhou, and Yiwu regions of Zhejiang Province, the chickens are raised in chicken coops and outdoor free range. The fecal samples were collected (3–5 g for each sample) from the 4 corners and the central area of the farms using disposable sterile PE gloves, and the sample number, collection time, location, and age of chickens were recorded carefully, and then the samples were brought back to the laboratory for examination of Eimeria. spp. The feces samples were analyzed by the traditional flotation method using saturated saline. Briefly, 1 g feces were weighted and put in a beaker, the samples were ground with a glass stirring rod with 30 mL saturated saline, then a 60-mesh sieve was used to filter the feces residue. Subsequently, the filtrate was added to a modified McMaster counting plate, and the coccidial oocysts were observed using the microscope (10 ×). The oocysts per gram feces (OPG) was calculated (the maximum, minimum and average OPG), and triplicates observation for each sample were performed to ensure accurate results.

Isolation of the Eimeira tenella Yiwu isolate

The cecum of the sick or dead chicken was collected and cut into pieces, then soaked in sterile distilled water for 2 to 3 h, the soaking liquid was passed through 100-mesh and 200-mesh copper sieves for 3 times. Subsequently, the filtered liquid was transferred into a sterilization bottle and centrifuged at 4,000 rpm/min for 5 min. The precipitation was suspended using triple volume of saturated saline and was centrifuged at 4,000 rpm/min for 5 min, and then the oocysts were collected and washed with 10 times volume of distilled water. After centrifugation, the supernatant was discarded slowly and the Eimeria oocysts were sporulated at 28°C (200 rpm) using a 2.5% (w/v) aqueous potassium dichromate solution, and then the sporulated oocysts and sporocysts were examined at 400 × magnification. Finally, the infective sporulated oocysts were stored at 4°C until used.

Ten 14-day-old SPF chickens were inoculated with sporulated oocysts (5 × 105). The survived chickens were slaughtered 8 days after challenge and cecal contents were harvested to obtain fresh oocysts. Then the E. tenella oocysts (about 100 oocysts) were collected according to the single oocyst separation technique as previous describe (Yim et al., 2011) with a minor modification. Briefly, 20 sporulated E. tenella oocysts were isolated from the sample using a micromanipulator and were kept in phosphate-buffered saline (PBS). Fourteen-days SPF chickens were infected with sporulated E. tenella oocysts, the fresh feces (4–11 d after infection) were collected and the E. tenella oocysts were identified using saturated saline floatation method and microscope observation.

Pathogenicity of the E. tenella Yiwu Isolate

SPF chickens (1-day-old) were raised until 21-day-old, and 25 coccidia-free broilers with similar weight were selected. The chickens were randomly divided into 5 groups (5 chickens/each group), among them, chickens in groups 1, 2, 3, and 4 were orally infected with diluted E. tenella, the inoculation doses were 5 × 105, 5 × 104, 5 × 103, and 5 × 102 successively, the blank control group was established. The growth status, forage, diarrhea, or bloody stools of each group of chickens were observed and the body weight of each chicken was also recorded 8 days postinfection. The feces were collected on the sixth to eighth day after infection and the OPG of each group was calculated according to McMaster's counting method, then the total number of oocysts per group was calculated. Finally, the pathogenicity of E. tenella was determined by clinical symptoms, body mass and oocyst excretion.

Relativeweightgainrate(%)=(theaverageweightgainofeachinfectiongroup/theaverageweightgainofun−infectedcontrolgroup)×100%.
Averageweightgainrate(%)=(thefinalweightafterinfectedwithEimeria−theinitialweightbeforeinfectedwithEimeria)/thenumberofchickensforeachgroup×100%.
Survivalrate(%)=(thenumberofsurvivingchickens/thetotalnumberofchickensfromthesamegroup)×100%.
Mortalityrate(%)=(thenumberofchickensthatdiedafterinoculationwithcoccidia/thetotalnumberofchickensinthesamegroup)×100%.

Experimental Design and Infection

The E. tenella isolate was tested for its sensitivity (Chapman, 1998) to 6 coccidiostats including sulfachlorpromazine sodium (1 g per kg feed), amproline (0.1 g per kg feed), toltrazuril (0.25 g per kg feed), salinomycin (0.65 g per kg feed), clopyridine (0.125 g per kg feed) and nicarbazine (0.125 g peer feed). The coccidiostats above were widely used in the investigated chicken farms. Briefly, 120 SPF chickens (14-day-old) were weighted and randomly divided into 8 groups (15 chickens/each group), all of the chickens have almost the similar weights. Then the chickens were administered with various coccidiostats beginning 2 d before infection with 5 × 104 sporulated oocysts, and these coccidiostats were offered throughout the entire experimental period. At the same time, the uninfected control group (N-ctl) received distilled water and the untreated infected group (P-ctl) was given the respective sporulated oocysts. The clinical signs were monitored daily and the survival rate, body weight gain, lesion scores, oocyst value and anticoccidial index (ACI) were recorded and measured.

Drug Resistance of the E. tenella Yiwu Isolate

The drug resistance was evaluated according to the (POAA), ACI, (RLS) and (ROP). ACI = (rate of relative body weight gain + survival rate) – (lesion score + oocyst value). Oocyst value = (fecal oocyst number of treated-infected group) / (fecal oocyst number of P-ctl) × 100%. An ACI value ≥ 160 indicated sensitivity and an ACI value < 160 represented resistance (Johnson and Reid, 1970). Growth and survival ratio (GSR) = (the final weight including the cage weight) / (the initial weight including the cage weight). POAA = (GSR in medicated group – GSR in infected-unmedicated group) / (GSR in uninfected-unmediated group – GSR in GSR in infected-unmedicated group) × 100%. A POAA value ≥ 50% indicated sensitivity and a POAA value < 50% indicated resistance (Arabkhazaeli et al., 2013). RLS = (mean lesion score of infected-unmedicated group–mean lesion score of treated-infected group) / (mean lesion score of uninfected-unmediated group) × 100%. A RLS value ≥ 50% indicated sensitive and a RLS value < 50% indicated resistance. ROP = (average oocyst output in treated-infected group) / (average oocyst output in infected-unmedicated group) × 100%. ROP ≥ 15% was judged to be sensitive and ROP < 15% was resistance (Lan et al., 2017).

Statistical Analysis

The statistical analysis was performed using SPSS Statistics 25 Software or GraphPad Prism 5. Differences in groups were compared by Chi-square test, and P value of < 0.05 was considered significant difference.

RESULTS

Prevalence of Eimeria spp. in Partial Regions of Zhejiang Province

A total of 600 fecal samples were collected randomly from 12 farms of 4 regions (Li shui, Taizhou, Hangzhou, and Yiwu) of Zhejiang province, China. Among the 600 examined samples, 325 (54.2%) samples were tested positive for Eimeria oocysts (Table 1). The prevalence of Eimeria oocysts were ranged from 18 to 100% in these 12 farms. Chickens under 40 d of age have the highest infection rate (97.5%), which was significantly higher (P < 0.01) than that in chickens between 60 and 85-days-old (40.5%) and chickens over 90-days-old (24.5%). The average OPG was 16,602 in chickens < 40 d, 3,530 in chickens between 60 and 85 d and 1,040 in chickens >90 d (Table 2). Among all the samples, the chickens from Taizhou and Yiwu were mainly raised intensively on the ground with litter, and the coccidian infection rate was 82.8% (207/250), which was significantly higher than that in Lishui and Hangzhou (33.7%, 118/350) (P < 0.05).

Table 1.

Prevalence of Eimeria infection in chicken in 4 regions of Zhejiang province, China.

Regions Age (d) No. of examined (%)1 No. of positive Prevalence rate (%) Feeding method
Lishui farm A 120 50 9 (1.5) 18.0 Free range
Lishui farm B 85 50 21 (3.5) 42.0 Free range
Lishui farm C 82 50 22 (3.7) 44.0 Free range
Taizhou farm A 36 50 47 (7.8) 94.0 Ground captivity
Taizhou farm B 36 50 50 (8.3) 100.0 Ground captivity
Taizhou farm C 37 50 50 (8.3) 100.0 Ground captivity
Hangzhou farm A 70 50 20 (3.3) 40.0 Free range
Hangzhou farm B 60 50 18 (3.0) 36.0 Free range
Hangzhou farm C 90 50 12 (2.0) 24.0 Free range
Hangzhou farm D 150 50 16 (2.7) 32.0 Free range
Yiwu farm A 32 50 48 (8.0) 96.0 Ground captivity
Yiwu farm B 120 50 12 (2.0) 24.0 Ground captivity
Total 600 325 (54.2) 54.2
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1

The percent accounts for the total chickens.

Table 2.

Chickens coccidian infection in different ages and feeding models.

Ages (d) No. of examined (%) No. of positive (%) Prevalence rate (%) OPG (range) OPG (average)
< 40 d 200 195 (32.5) 97.5 0−536,300 16,602
60–85 d 200 81 (13.5) 40.5 0−43,500 3,530
90–150 d 200 49 (8.2) 24.5 0−1,700 1,040
Total 600 325 54.2
Feeding method
Free range 350 118 (19.7) 33.7
Ground captivity 250 207 (34.5) 82.8
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Abbreviation: OPG, oocysts per gram of feces.

Pathogenicity of the E. tenella Yiwu Isolate

Four doses of the E. tenella isolate (5 × 105, 5 × 104, 5 × 103, and 5 × 102) were used to analyze the pathogenicity. The clinical symptoms can be observed in all of the infection chickens, such as decreased water and food intake, decreased fecal output. Among the 4 infection groups, all of the chickens infected with 5 × 105 E. tenella died on the seventh day after the infection. In the 5 × 104 dose infection group, 2 chickens died on the fifth and sixth day after the infection, and the mortality rate was 40% (2/5). Only 1 chicken died for the 5 × 103 dose infection group, and no chickens died for group 4 (5 × 102 dose) and 5 (control) (Figure 1A).

Figure 1.

Figure 1

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Assessment of survival curves and body weight gain after infected with E. tenella. Survival rate of chickens after challenge with 102, 103, 104, and 105 sporulated oocysts of E. tenella. Chickens uninfected were as blank control (A). Survival rate was monitored daily after infected with E. tenella from 4 to 8 d after challenge. The body weight gain was calculated after infection with different doses of sporulated oocysts (B). *p < 0.05, **p < 0.01.

The bloody stool score (Johnson and Reid, 1970) and average lesion score of chickens from group 1 (5 × 105), group 2 (5 × 104), group 3 (5 × 103), and group 4 (5 × 102) decreased successively (Figure 2 A–J), and the average weight gain (g) and relative weight gain (%) increased successively. As shown in Table 3, the average weight gains of group 1 and 2 were significantly lower than that in the uninfected- unmedicated negative control (group 5) (P < 0.01), and chickens infected with 5 × 103 and 5 × 102 dose of E. tenella also showed lower weight gain compared to the negative control (P < 0.05). The present results also indicated that the weight gains of the low-dose E. tenella infection groups (5 × 103 and 5 × 102) were higher than the other 2 groups (5 × 105 and 5 × 104) (P < 0.05) (Figure 1B).

Figure 2.

Figure 2

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Evaluation of blood stool and intestinal lesions after challenge of E. tenella. Blood stool from the chickens infected with 102, 103, 104, and 105 sporulated oocysts of E. tenella (A–D), uninfected negative control group (E). Intestinal lesions from the chickens infected with 102, 103, 104, and 105 sporulated oocysts of E. tenella (F–I), negative control group (J).

Table 3.

Weight gain and pathological changes of experimental chickens.

Group Inoculation dosage Average weight gain (g) Relative weight gain rate (%) Bloody stool score Lesion score Mortality rate (%)
1 5 × 105 34.0 ± 32.8a,⁎⁎ 18.1 2.5 4 100
2 5 × 104 69.4 ± 45.2a,⁎⁎ 36.8 1.5 3.5 40
3 5 × 103 100.4 ± 24.8b,* 53.2 0.8 2 20
4 5 × 102 116.4 ± 30.7b,* 61.7 0.3 1 0
5 0 188.8 ± 82.4 c — 0 0 0
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a,b,c

The same superscripts in a column of data indicate that the difference is not significant (P > 0.05).

⁎

P < 0.05 vs. uninfected- unmedicated negative control (N-ctl),

⁎⁎

P < 0.01 vs. N-ctl.

Evaluation of Anticoccidial Effects of 6 Coccidiostats

All the chickens survived in the groups infected with the Yiwu isolate of E. tenella and fed with the anticoccidial drugs. The body weight gain, lesion scores, oocyst value and ACI value were shown in Table 4. At the present results, sulfachloropyrazine sodium and toltrazuril can effectively treat the chickens infected with Yiwu isolate, while the isolate performed resistance to amproline, clopidol, salinomycin, and nicarbazine. The initial body weight, final body weight, GSR, and POAA were presented in Table 5, chickens from sulfachloropyrazine sodium, toltrazuril, clopidol, salinomycinand, and nicarbazine infected-medicated groups showed significantly higher final body weight compared with that in infected-unmedicated positive control (P < 0.05). Then all of the POAA were calculated according to the GSR. The results indicated that the POAA of amproline < 50%, which indicated that the E. tenella Yiwu isolate has developed resistance to amproline. In the present study, RLS and ROP were recorded in Table 6. We found that the Yiwu isolate has performed resistance to toltrazuril, clopidol, salinomycin, and nicarbazine according to RLS, and this isolate exhibited relatively severe resistance to drugs of salinomycin and nicarbazine according to the ROP values.

Table 4.

Anticoccidial index of 6 coccidiostats.

Drugs Survival rate (100%) Body weight gain rate (%) Lesion scores Oocyst value Anticoccidial index (ACI)
SC 100 79.8 14 5 160.8
AM 100 49.7 15 5 129.7
TOL 100 92.6 22 5 165.6
CL 100 75.7 20 5 150.7
SA 100 92.2 25 10 157.2
NI 100 77.6 22 5 150.6
P-ctl 100 47.7 30 40 77.7
N-ctl 100 100 0 0 200
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Abbreviations: AM, amproline; CL, clopidol; N-ctl, uninfected-unmedicated negative control; NI, nicarbazine; P-ctl, infected-unmedicated positive control; SA, salinomycin; SC, sulfachloropyrazine sodium; TOL, toltrazuril.

Table 5.

Weight gains and percent of optimum anticoccidial activity.

Drugs1 Initial body weight (g) Final body weight (g) GSR Percent of optimum anticoccidial activity (POAA, %)
SC 64.2±6.20 101.4±17.5 a 1.58 60.1
AM 63.6±7.54 87.0±11.9 b 1.37 5.30
TOL 62.5±8.26 106.6±11.0 a 1.71 94.7
CL 64.4±5.42 100.3±21.29 a 1.56 55.3
SA 62.8±10.30 106.5±20.9 a 1.70 92.1
NI 66.2±6.79 103.2±15.1 a 1.56 55.3
P-ctl 64.8±4.26 87.4±17.9 b 1.35 /
N-ctl 65.3±6.52 112.7±14.7 a 1.73 /
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1

AM, amproline; CL, clopidol; GSR, the final weight including the cage weight ÷ the initial weight including the cage weight; NI, Nicarbazine; P-ctl, infected-unmedicated positive control; SA, Salinomycin; SC, sulfachloropyrazine sodium; TOL, toltrazuril.

a,b

The same superscripts mean no difference, the different superscripts represent significant differences (P < 0.05).

Table 6.

Results of average lesion scores/reduction of lesion scores and the average oocyst production/relative oocyst production.

Drugs Average lesion scores Reduction of lesion scores (%) Average oocyst production (107) Relative oocyst production (%) Oocyst value
SC 1.4 53.3 22.8 9.4 5
AM 1.5 50.0 19.6 8.1 5
TOL 2.2 26.6 16.2 6.7 5
CL 2.0 33.3 23.7 9.8 5
SA 2.5 16.6 114 47.1 10
NI 2.2 26.6 41.4 17.1 5
P-ctl 3.0 / 241.7 / 40
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Abbreviations: AM, amproline; CL, clopidol; NI, nicarbazine; P-ctl, infected-unmedicated positive control; SA, salinomycin; SC, sulfachloropyrazine sodium; TOL, toltrazuril.

Synthetic Judge of Drug Resistance for the E. tenella Yiwu Isolate

According to the results of ACI,POAA,RLS, and ROP, the isolate has developed severe resistance to drugs of salinomycin and nicarbazine, moderate resistance to drugs amproline and clopidol, slight resistance to toltrazuril and this isolate showed no resistance to sulfachloropyrazine sodium Table 7.

Table 7.

Comprehensive determination of drug resistance of Eimeria tenella isolates.

Drugs1 ROP (%) RLS (%) POAA (%) ACI Resistance2
SC 9.4 - 53.3 - 60.1 - 160.8 - None
AM 8.1 - 50.0 - 5.30 + 129.7 + Moderate
TOL 6.7 - 26.6 + 94.7 - 165.6 - Slight
CL 9.8 - 33.3 + 55.3 - 150.7 + Moderate
SA 47.1 + 16.6 + 92.1 - 157.2 + Severe
NI 17.1 + 26.6 + 55.3 - 150.6 + Severe
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1

AM: amproline; SC: sulfachloropyrazine sodium; TOL: toltrazuril; CL: clopidol; SA: salinomycin; NI: nicarbazine.

2

Severe drug resistance (+++ / ++++), moderate drug resistance (++), slight drug resistance (+) and none + meant no drug resistance.

DISCUSSION

Coccidian infection in chickens has been reported worldwide, and chickens of all different ages can be infected. Avian coccidiosis poses a significant challenge to the poultry industry (Ogedengbe et al., 2011). In the present study, 54.2 % (325/600) of the samples tested positive for Eimeria spp., which was consistent with the finding of other authors (Debbou-Iouknane et al., 2018), the mixed infection rate with Eimeria spp. was 54.28%. Our result was lower than the 87.72% found in Anhui province, central China (Huang et al., 2017), 97.17 % in Hubei and Hunan provinces, central China (Geng et al., 2021), 96% in the southern region of Brazil (Moraes et al., 2015), and 78.7% in Korea (Lee et al., 2010). However, the prevalence rate of chicken coccidia in the present study was higher than that recorded in the previous researchs in Zhejiang province in China (30.7%) (Lan et al., 2017), 31.5% in Khuzestan, southwest Iran (Hamidinejat et al., 2010). Eimeria infections in chickens are present globally, however, the infection rates vary widely, which maybe related to the breeding density of the chickens, the immune statue of the animals, feeding and management conditions, breeding methods of the chicken, age, climate, size of flock, etc (Shirzad et al., 2011). Age is a key factor in determining infection rates of coccidia, younger animals are more susceptible to natural infections than older ones (Chengat Prakashbabu et al., 2017), which has been attributed to a more development immune system in adults than in younger chickens (Taylor et al., 2003). In the present study, the prevalence rates of Eimeria spp. were ranged from 18 to 100% in these 12 farms. The infection rate of coccidia in chickens under 40 d was significantly higher (P < 0.01) than that in chickens between 60 and 85-days-old (40.5%) and chickens over 90-days-old (24.5%), while no significant difference was found in chickens over 60 d (60–85 d compared with group over 90 d) (P > 0.05). Our results recorded a rate almost similar to the results of the previous study which reported a higher occurrence can be seen in the chickens ranging of 5 to 6 weeks (Shirzad et al., 2011). In another study conducted in Bejaia province of Algeria, higher prevalence of coccidiosis was found at the age of 32 to 46 d (Debbou-Iouknane et al., 2018), the prevalence was higher in younger chickens (60%) compared with older ones (37%) (Bachaya et al., 2012). Actually, many reasons affect the infection, such as various predominant Eimeria spp., different anticoccidial drugs and injection procedures and dosage. In the present study, the chickens from Taizhou and Yiwu were mainly raised intensively on the ground with litter, and the infection rate of coccidia was 82.8% (207/250), which was significantly higher than that in Lishui and Hangzhou (33.7%, 118/350) (P < 0.05). The scales of farms in Taizhou and Yiwu are larger than farms in Lishui and Hangzhou. A survey of subclinical coccidiosis in broiler showed that large-sized farms had more coccidiosis than small-sized farms because of more water, feed, and litter are needed (Reza Razmi and Ali Kalideri, 2000), then these could produce large amounts of feces, so poor feeding management can easily increase the infection of coccidia. Braunius (1980) also showed that chickens in larger farms were more alarming than those from the smaller farms (Braunius, 1980).

E. tenella is one of the most harmful species because of its wide prevalence and high pathogenicity (Witcombe and Smith, 2014), and intestinal coccidiosis in chickens causes enormous economic losses. The main criteria for determining the pathogenicity of chickens coccidiosis include lesion score, oocysts output, body weight, and the survival rate (McManus et al., 1968).

In the present study, an E. tenella Yiwu strain was isolated, the pathogenicity results showed that all of the chickens infected with 5 × 105 E. tenella died on the seventh day of infection. The bloody stool scores and average lesion scores decreased successively for the groups infected with E. tenella (5 × 105, 5 × 104, 5 × 103, and 5 × 102) and the average weight gain (g) and relative weight gain (%) increased successively. The mortality rates were 100%, 40% and 20% respectively in 5 × 105, 5 × 104, 5 × 103 infection groups in our results. It was previously (Abu-Akkada and Awad, 2012) demonstrated that the mortality rate of E. tenella infection ranged 5.6 to 22.2% when the chickens infected with 5 × 103 sporulated oocysts. Taylor et al (2003) found that not all experimental groups of chickens showed signs of death, and the survival rate of some pathogenic E. tenella isolates ranged from 70 to 90% (the infection dose was 5  ×  104) (Taylor et al., 2003). Although inoculation with the same dose of coccidial oocysts, the mortality rate will vary depending on the pathogenicity and virulence of the particular strain (Abu-Akkada and Awad, 2012), and the ages, breeds of chicken for example, often affect the coccidia pathogenicity (Chengat Prakashbabu et al., 2017).

Currently, supplement of drugs in feed or water remains the most widely used method to control coccidiosis (Chapman et al., 2010), the large-scale and long-term use of anticoccidial drugs lead to the emergence of drug-resistant strains (Chapman et al., 2010), several resistant strains have been reported (Mattiello et al., 2000; Peek and Landman, 2003) and resistance appears to be widespread. Our experiments confirmed that 5 drugs treatment groups (sulfachloropyrazine sodium, toltrazuril, clopidol, salinomycinand, and nicarbazine) showed significantly higher body weight gain compared with that in infected-unmedicated positive control (P < 0.05). The Eimeria Yiwu isolate has developed resistance to amproline (POAA < 50%). According to the RLS values, we found the E. tenella Yiwu isolate has performed resistance to toltrazuril, clopidol, salinomycin, and nicarbazine and this isolate exhibited relatively severe resistance to drugs of salinomycin and nicarbazine from the ROP values. In general, the E. tenella Yiwu isolate showed slight resistance to toltrazuril, moderate resistance to amproline and clopidol, severe resistance to salinomycin and nicarbazine, but this isolate was sensitive to sulfachloropyrazine sodium. Compared with a previous study, which found that QZ isolate (Quzhou, Zhejiang, China) developed severe resistance to sulfachloropyrazine, toltrazuril and amprolium, while another HZ isolate (Hangzhou, Zhejiang, China) developed moderate resistance to nicarbazin, and severe resistance to sulfachloropyrazine, toltrazuril, and amprolium (Lan et al., 2017). Our results are in agreement with similar study conducted in Nigeria (Ojimelukwe et al., 2018), and all isolates showed reduced sensitivity to salinomycin (Arabkhazaeli et al., 2013). Chapman and Hacker (1994) recorded that the Eimeria field isolates developed reduced sensitivity to clopidol (Chapman and Hacker, 1994). There are differences between various isolates in response to the same or similar anticoccidial drugs.

Based on the current serious drug resistance of Eimeria spp., there is an urgent need for new approaches to prevent coccidian infection in chickens. Studies have indicated that edible herbs and natural products may be acceptable for consumers and plants performed better anticoccidial activities in some researches (Wunderlich et al., 2014; Daneshmand et al., 2021; Mohamed et al., 2021). It was reported that polyphenols, flavonoids, polysaccharides, and other natural products come from plants could disturb the balance of oxidants and antioxidants on Eimeria oocyst (Molan et al., 2009; Hamilton et al., 2020). Although only a few plants and their natural products showed similar anticoccidial activity, they may have broad application prospects in future (Daneshmand et al., 2021).

In conclusion, the present study indicated that 54.2 % (325/600) of the examined chickens were infected with Eimeria spp., the coccidial infection rate in younger chickens (< 40 d) was significantly higher (P < 0.01) than that in older chickens (60–90 d). Chickens in large-scale farms performed higher prevalence than in small-scale farms. Then, an E. tenella Yiwu strain was isolated successfully and the pathogenicity of this isolate was analyzed. The results showed that the mortality rate of E. tenella infection ranged 20 to 100% when the chickens infected with 5 × 103, 5 × 104, and 5 × 105 sporulated oocysts successively, and all of the chickens survived when infected with 5 × 102 oocysts. According to the ACI, POAA, RLS, and ROP values, we found that the E. tenella Yiwu isolate showed slight resistance to toltrazuril, moderate resistance to amproline and clopidol, severe resistance to salinomycin and nicarbazine, but this isolate was sensitive to sulfachloropyrazine sodium. Considering the severe drug resistance in poultry industries, there is an urgent need for new anticoccidial strategies and alternative approaches to prevent and treat coccidiosis in chickens.

ACKNOWLEDGEMENTS

This work was supported by grants from the National Natural Science Foundation of China (No. 32072883, 31802183), Hangzhou Agricultural and Social Development Project（202203B19), Zhejiang Province “Sannongjiufang” Science and Technology Cooperation Project (No. 2023SNJFF058), Hangzhou Science and Technology Commissioner Project (20221122I30), and International Cooperation Projects of Zhejiang Academy of Agricultural Sciences.

DISCLOSURES

All of the authors have no personal financial interests or professional relationships related to the subject.

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