Prevalence of Eimeria parasites and sulfachloropyrazine sodium resistance in chicken farms in the Hubei and Henan provinces

Background Coccidiosis is an intestinal parasitic disease that causes huge economic losses to the poultry industry globally. At present, the primary control strategy is administration of anticoccidial drugs with feed. However, overuse of anticoccidials, such as sulfachloropyrazine sodium (SC), has resulted in an increase in the emergence of drug resistance. Methods: We aimed to evaluate coccidiosis prevalence and SC resistance in field isolates to provide reasonable guidance on the use of anticoccidial drugs in the Hubei and Henan provinces. We collected 318 fresh fecal samples from 137 chicken farms. We used internal transcribed spacer 1 (ITS1) sequence of ribosomal DNA to identify the species from 94 samples that were collected from different farms and to assess drug resistance. Results: As shown by genus-specific PCR results, the positivity rate of Eimeria was 97.17 % (309/318), and the most common species were E. mitis (66.67%), E. tenella (46.86%), and E. necatrix (41.51%). Animal experiment demonstrated that 25 strains were completely resistant to SC, among which 16 were from Henan and nine were from Hubei. Twenty-four strains were partially resistant, among which 8 and 16 strains were identified from Hubei and Henan, respectively. Conclusions: In summary, these data indicated that chicken coccidia is ubiquitous and SC resistance is widespread, in the Hubei and Henan provinces. The results provide important insights into the control of chicken coccidiosis in this region.

Furthermore, nine different Eimeria spp. are described [1], and seven species of Eimeria are mainly recognized worldwide [2]. Each species of Eimeria infects chicken with absolute host specificity [1], and the virulence of coccidia varies with the type of coccidiosis [1], E. necatrix is recognized as the most pathogenic species [3].
Coccidiosis causes huge economic losses to the global poultry industry [4].
According to incomplete statistics, the annual economic burden of the prevention and treatment of chicken coccidiosis in the world exceeds 3 billion USD [4]. In the past 30 years, the poultry industry in Asia has developed extremely rapidly, and China as Asia's largest economy has become the third largest chicken-producing country in the world [5]; the annual burden of chicken coccidiosis exceeds 73 million USD [5], accounting for approximately 30% of all chicken disease burden [5].
As such, it is extremely important to understand the detection and control of pathogens. The traditional pathogen classification of avian coccidiosis depends on features such as morphology and life history. At present, PCR is used for the classification and identification of parasites and for studying genetic evolution. In the case of coccidiosis caused by Eimeria, the 5' end of the ITS1 region is widely used for species identification [6,7]. Lew et al. amplified and cloned ITS-1 of five species of Eimeria, namely E. tenella, E. necatrix, E. acervulina, E. brunetti, and E. mitis, isolated from Australia using PCR [6]. Hamidinejat et al. identified Eimeria spp. in commercial broilers using PCR, based on ITS1 regions of rDNA and showed that the most prevalent species in Khuzestan was E. tenella [ 8].
So far, the control of chicken coccidiosis still relies on the addition of coccidiostats to feed [9]. Coccidiostats mainly include chemical synthetic anticoccidial drugs and polyether ionophore antibiotic anticoccidial drugs [10]. Sulfonamides are an important and commonly used synthetic chemical drugs. They mainly achieve anticoccidial effect by inhibiting the growth and development of coccidia; they have no direct anticoccidial effect. Aminobenzoic acid is one of the most important raw materials in the synthesis of dihydrofolate. The chemical structure of sulfonamide is similar to that of p-aminobenzoic acid. The latter can compete with aminobenzoic acid for dihydrofolate synthetase, thereby hindering the formation of dihydrofolate, affecting the synthesis of nucleic acids, inhibiting the growth and reproduction of coccidia, and achieving anticoccidial effects [11,12].
In this study, we collected fresh fecal samples from chicken farms in different areas of Hubei and Henan provinces, investigated anticoccidial drugs in chicken farms, and analyzed SC resistance of Eimeria. The results of this study have great significance for the prevention and treatment of avian coccidiosis in the region.

Fecal sample collection
Data on age, variety of birds, and the method of breeding, size of the chickens on the farm, the occurrence of coccidiosis, and the history of drug use were collected across the Hubei and Henan provinces. Fecal samples were collected from 137 different local chickens' farms (commercial broiler production farms and backyard flocks) between June 2017 and October 2018 (Additional file 1: Table S1). No less than 500 g of sample was collected from each farm.
Eimeria oocysts obtained from the fecal samples were purified by saturated sodium nitrate flotation technique and sporulated using standard procedures [13]. The saturated sodium chloride floatation method was performed as follows. First, the 6 fecal sample was mashed and mixed with a right quantity of water to achieve suspension; the suspension was filtered through a 180 diameter mesh, and the obtained filtrate was centrifuged at 2000 to 2500 rpm/min. The supernatant was discarded; the obtained precipitate was mixed with saturated saline solution and centrifuged at 3000 rpm/min; Subsequently, we collected the supernatant, which was diluted five times with purified water and centrifuged at 3000-3500 rpm. The obtained precipitate was contained oocysts of Eimeria spp.

DNA extraction
For the extraction of coccidia sample DNA, the sporulated oocysts were washed three times with deionized water, and subsequently, the oocyst wall was vortexed three times using Lysing Beads-Matrix D lysate (MP Biomedicals, Shanghai, China) in a rapid sample preparation device to release sporozoites and 20 µl (20 mg/ml) of proteinase K (Genomic DNA Kit, TIANGEN, Beijing, China) was added. Subsequently, the mixture was incubated at 56 °C for 10 h, and DNA was extracted by using a Genomic DNA Kit (TIANGEN) according to the manufacturer's instructions.

Species identification of Eimeria
To confirm the species identity of these Eimeria isolates, seven pairs of speciesspecific primers (for E. acervulina, E. brunetti, E. mitis, E. necatrix, E. maxima, E. praecox, and E. tenella) that amplify ITS1 region were used. Sequences of the PCR primer pairs used were summarized in Table 1 [ 14,15,16]  Academy of Agriculture Sciences. The selected isolates were orally administered to 8 specific pathogens free (SPF) chickens (14-day-old). After 7 days of inoculation, the chickens were sacrificed, the oocysts were obtained from the intestinal contents by protease digestion method [17], the oocysts in the previous step were transferred to 2% w/v potassium dichromate in a 50 ml centrifuge tube, and placed in an incubator 28.6 ℃ for 4-7 days. If sporulation was identified in > 80% of the oocysts by microscopic examination, they were stored in 2.5% potassium dichromate at 4 ℃ [18].
Ninety-four isolates were randomly selected to evaluate drug effectiveness. Nine hundred and forty SPF chickens (14-day-old) were weighed and randomly divided into 94 groups; each group included 8-12 chickens, 4-6 of which were treated with 2 g/kg SC in feed. Every chicken was inoculated with 1 ml of 5 × 10 4 sporulated oocysts. The blank control group consisted consisting of 10 chickens, which were only fed deionized water. Chickens were weighed at 21 days of age, and subsequently, the lesional scores were recorded. From each post-infection group between 5-7 days, single droppings of chicken feces were collected each day to evaluate the relative number of oocysts per gram of feces (OPG) [17]. The coccidial lesions present in the chickens were scored on a 4-point scale (0-4 points), as described previously [19]. OPG values were determined using the McMaster's method [20]. The relative oocyst production rate (OPR) is equal to the ratio of the oocysts produced by the infected-treated group versus those produced by the infected-untreated group. Subsequently, the anticoccidial index (ACI) was calculated to assess drug effectiveness [21].

Results
Identification of Eimeria species in field isolate 9 A total of 318 samples were collected from chicken farms in the Hubei and Henan provinces (Fig. 1). Each isolate was amplified with seven pairs of species-specific primers (Fig. 2). The specific identification results of each sample are shown in

Huanggang, and Wuhan) and southwestern (Enshi Tujia and Miao Autonomous
Prefecture and Yichang) Hubei (Fig. 3a). E. tenella was not detected in Kaifeng, Jiaozuo, Nanyang, and Shangqiu of the Henan Province (Fig. 3b) and Xiangyang of the Hubei province (Fig. 3c). Only the chicken feces samples from Yichang and Wuhan in the Hubei province did not show E. mitis (Fig. 3c). Additionally, we found that E. maxima was detected only in Sanmenxia of the Henan Province (Fig. 3b) and Wuhan, Huanggang, Suizhou, Xiaogan, Yichang and Enshi Tujia, and Miao Autonomous Prefecture in the Hubei province (Fig. 3c).
Mixed-species infections were common in the Hubei and Henan Provinces (Fig. 4a).
The single Eimeria infection rate was lower in the Hubei province (15.44%) than in the Henan province (34.07%) (Fig. 4b) (Fig. 4b), especially in Suizhou and Huanggang (Fig. 4a). Therefore, it could be preliminary indicated that the Hubei province showed a higher proportion of mixed infections than the Henan province did.
Mixed-species infections were common irrespective of the type of the chicken;  (Fig. 4c).

Sulfachloropyrazine sodium (SC) sensitivity of Eimeria
In this study, among the 94 isolates selected (Table S3), 53 were from the Henan province and 41 were from the Hubei province. We found that 25 of the isolates-16 Henan and nine from Hubei-had an ACI of less than 160, indicating that these strains were fully resistant. ACI of 24 strains-16 Henan and eight from Hubei-was 160-180, indicating that these strains were partially resistant. The ACI of the other strains was > 180, indicating that these strains were not resistant (Table 3). Geographically, we found that the sulfa-resistant Eimeria spp. were distributed in the central (Zhengzhou, Kaifeng, Xuchang, Luohe, and Zhumadian) and western (Sanmenxia, Luoyang, and Jiyuan) parts of Henan (Fig. 5). In Hubei, it was distributed in the northwest (Shiyan and Shennongjia Forest Area) and southeast (Xiantao, Xianning, and Huangshi).

Discussion
In this study, 97.17% of the samples tested positive to for Eimeria. The high prevalence of Eimeria in field samples was relatively consistent with the percentages recorded previously in France (95%) [22]; however, these values are higher than those recorded in the Anhui Province in China (87.7%) [5], north India (81.3%) [23], and Korea (78.7%) [24]. This could be possibly explained by differences in detection methods and geographical conditions. In addition, most of the samples come from small chicken farms, which have high stocking densities and are commonly populated with specialized, genetically homogeneous breeds that are possibly conducive to disease transmission. in Henan province (Table 2); however, Eimeria infections in Hubei were more complex than those in Henan (Fig. 4b). Indigenous chickens showed a lower relative abundance of Eimeria species than the others (Fig. 4c). The reasons for this phenomenon must be investigated more comprehensively in the future. showed that the field isolates showing drug resistance in the Henan province were higher than those in the Hubei province; therefore, it could be preliminarily indicated that SC resistance of chicken coccidia in Henan was more common than that in the Hubei province. Possible explanation for this is that most of the samples come from semi-intensive small chicken farms in the Henan province and the use of anticoccidial drugs was not standardized by technical guidance. Surprisingly, by contrast, the results were different from those reported in Anhui Province in China by Huang et al. [5], and many of the strains were not resistant (ACI ≥ 180), most likely because reducing the use of sulfonamides resulted in expanding the population of sulfa drug-sensitive coccidia strains; this may explain why the phenomenon of drug resistance was relatively improved in the Hubei and Henan provinces.

Conclusions 14
In conclusion, firstly, this study completed an epidemiological survey of coccidiosis Relative oocyst production rate.
Declarations Figure 2 ITS1-specific primers for the identification of Eimeria species. Abbreviations: E.a, Eimeria ace Map indicating the number and type of ACI selected from Henan and Hubei Provinces, China.