Development of a qPCR to diagnose the genus Eimeria in sheep and goats

doi:10.21203/rs.3.rs-1619199/v1

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Development of a qPCR to diagnose the genus Eimeria in sheep and goats

https://doi.org/10.21203/rs.3.rs-1619199/v1

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Coccidiosis of sheep and goats is caused by protozoa in the genus Eimeria. These protozoa mainly affect young animals, causing a decrease in production and consequent economic losses. Routine diagnosis is made through morphological observation of the oocysts, which has several limitations. The objective of the present study was to develop a real-time PCR (qPCR) technique for the diagnosis of Eimeria spp. in sheep and goats. For this purpose, the 18S rRNA region of the DNA of these parasites was selected because it is a region with low variability among Eimeria spp. The qPCR technique was developed using SYBR Green, resulting in a PCR with high sensitivity, and the ability to amplify samples containing only one oocyst of an Eimeria spp. There was no cross-reaction with other intestinal protozoa, such as Blastocystis, Microsporidia, Cryptosporidium, and Giardia duodenum. The repeatability test showed that the coefficient of variation was less than 2%. This indicated that this method has good sensitivity, specificity, and reproducibility. Thus, the feasibility of using qPCR in the diagnosis of the genus Eimeria was demonstrated in this study. This technique was less laborious and required less skill and diagnostic training compared with the micromorphometry technique conventionally used for this diagnosis.

18S rRNA

Sheep

Goat

Coccidiosis

Eimeria

qPCR

Coccidiosis is an important parasitic disease of ruminant livestock caused by protozoan parasites of the genus Eimeria, with lambs and kids between 1 and 6 months of age being most susceptible (Bangoura and Bardsley 2020). Eimeria are host specific, meaning that an Eimeria species that infect goats does not infect sheep, and vice versa (Nicole and Navarre 2018).

Thus far, 12 intestinal and one abomasal Eimeria species have been observed in sheep (Ammar et al. 2019; Dittmar et al. 2010), with 10 intestinal species recently found in goats (Das et al. 2017; Silva et al. 2014). Eimeria ovinoidalis is the most pathogenic species in sheep. Among those found in goats, Eimeria ninakohlyakimovae and Eimeria caprina are the most pathogenic species (Bangoura and Bardsley 2020). Others are less pathogenic or non-pathogenic. Although some species are not pathogenic, as parasites, they absorb nutrients from hosts, which could be responsible for the decrease in yield seen in infected animals.

The occurrence of Eimeria spp. varies among countries from 48.7 to 100% (Balicka-Ramisz et al. 2012; Cavalcante et al. 2012; Mohamaden et al. 2018; Saratsis et al. 2011). In China, the prevalence of Eimeria spp. in sheep and goats was 92.9% and 78.7%, respectively (Diao et al. 2022; Wang et al. 2010).

A few studies have developed PCR assays to diagnose Eimeria spp. For example, a primer was obtained for the genus Eimeria based on the 18S rRNA site, and was used to construct a phylogenetic tree comprising the main species in cattle (Kokuzawa et al. 2013). Yang et al. developed a qPCR technique using primers designed at the 18S rRNA region to quantify the production impacts of Eimeria in sheep (Yang et al. 2014).

Concerning technological advancements, new assays, such as real-time PCR (qPCR) are now available and capable of developing a more sensitive and specific diagnosis compared with that of conventional PCR. Hence, the goal of the present study was to develop and standardize a qPCR technique to diagnose Eimeria spp. in sheep and goats.

2.1. Samples

Positive fecal samples of Eimeria spp. were selected from naturally infected animals and stored in 2.0 mL microtubes at -20°C in the Veterinary Parasitology Laboratory of Henan Agricultural University.

Positive fecal samples of Giardia duodenalis, Blastocystis, Microsporidia, and Cryptosporidium parvum, detected by PCR, were used to evaluate the specificity of the primers developed in this study.

2.2. Primer design

The primers were designed from the 18S rRNA gene sequences of Eimeria spp. and deposited at GenBank with the following accession numbers: MW512853.1 (Eimeria sp. strain 20Q68-2), MN473507.1 (Eimeria parva isolate IQ-Deer No.36), MN473485.1 (E. parva isolate IQ-Deer No.14), MN473478.1 (E. parva isolate IQ-Deer No.7), MN473490.1 (Eimeria intricata isolate IQ-Deer No.19), LC507796.1 (Eimeria hirci), LC507795.1 (E. hirci), MW577425.1 (Eimeria arloingi strain 10), MW577427.1 (Eimeria christenseni strain 12), KX519412.1 (Eimeria ninakohlyakimovae), MW512852.1 (Eimeria sp. isolate 20Q68-1), MT337428.1 (Eimeria sp. isolate 5–12), MN149906.1 (Eimeria sp. isolate TW4), MT801036.1 (Eimeria sp. voucher SX2), MT801027.1 (Eimeria sp. voucher LX1), and MT801017.1 (Eimeria sp. voucher DJY4). After the selection of the sequences, the primers were aligned using the MEGA7 program (www.megasoftware.net). Following the alignment, a specific site was selected and used for primer design, which was performed with Primer Premier 5.0 (www.premierbiosoft.com/primerdesign). The primers obtained were named Eimeria 18S-F (5′-CAGGCTTGTCGCCCTGA-3′) and Eimeria 18S-R (5′-TTCGCAGTAGTTCGTCTTT-3′), with an amplicon of 168 bp.

2.3. DNA extraction

DNA extraction steps are performed in accordance with product instructions of the Stool DNA Kit (E.Z.N.A Stool DNA Kit, OMEGA, Bozeman, MT, USA).

2.4. Preparation of plasmid standard

Endpoint PCR was used with primer sets to obtain the PCR product of Eimeria spp. The reaction mixture (25 µL) contained 2.5 µL PCR buffer (10×), 2.0 µL dNTPs (2 mM each), 1.0 µL MgCl₂ (25 mM), 1.0 µL forward and reverse primers (10 µM), 0.2 µL DNA polymerase (1U·µL^− 1), 1.0 µL of genomic DNA purified from Eimeria spp., and 17.3 µL of PCR double-distilled water. PCR was carried out under the following conditions: an initial denaturation at 94°C for 4 min, followed by 35 cycles of 45 s at 94°C, 35 s at 55°C, and 1 min at 72°C. The final extension step was set for 7 min at 72°C. The PCR amplicon was purified from agarose gel, cloned into a pMD18-T vector, and transformed into Escherichia coli DH5α. Recombinant clones were selected by blue/white screening. The recombinant plasmid DNA was extracted using the SanPrep column plasmid DNA small amount extraction kit (Sangon Biotech, Shanghai, China) and sent to Shanghai Sangon Biotech for sequencing. Recombinant plasmids with the correct sequence of the Eimeria spp. amplicon (hereafter pMD18T-168) was used as the standard in qPCR.

2.5. Construction of standard curves for plasmid copy number determination

The pMD18T-168 plasmid was used to generate standard dilution series and the development of the qPCR test. The concentration of the plasmid standard solution was measured using NanoDrop 1000 (Thermo Fisher Scientific, Waltham, MA, USA), and the corresponding copy number was calculated. A tenfold dilution series of pMD18T-168, ranging from 3.43×10³ to 3.43×10⁸ copies/µL, was made and used to construct a standard curve. The reaction mixture contained 1.0 µL of plasmid DNA dilution, 1.0 µL Eimeria18S-F (500 nmol/L), 1.0 µL Eimeria18S-R (500 nmol/L), 10.0 µL of THUNDERBIRD® Next SYBR® qPCR Mix (TOYOBO, Osaka, Japan), and PCR double-distilled water to a final volume of 20 µL. An initial denaturation at 95°C for 30 s was followed by 40 cycles of 5 s at 95°C, 10 s at 56℃ and 15 s at 72°C. Threshold cycle (Ct) values in dilutions were measured in triplicate and plotted against the logarithm of their initial copy number. Each standard curve was generated by linear regression of the plotted points, and standard curve parameters were obtained. Ct was calculated under default settings for the qPCR soft system Software (ver. 4.0, Analytik).

2.6. Standardization of the qPCR

Each assay designed included negative and positive controls and the standard curve. The negative control was the PCR reaction without template DNA. The positive control was a PCR reaction containing guide (g)DNA of Eimeria spp. All controls and samples were assayed at least three times. The specificity of Eimeria spp. qPCR was evaluated by performing the method using gDNA from G. duodenalis, Blastocystis, Microsporidia, and C. parvum simultaneously. The analytical sensitivity (i.e., limit of detection) was established using eight replicates of serially diluted pMD18T-168 plasmid at 3.43×10⁷, 10⁶ 10⁵, 10⁴ 10³, 10², 10¹, and 10⁰ copies/reaction. gDNA was extracted from 10,000 coccidia oocysts, and diluted into five gradients of 10,000, 1,000, 100, 10, and 1 oocyst(s) according to the tenfold dilution series. The established qPCR was used to detect gDNA to determine the sensitivity of the method. The interassay precision of the qPCR was defined as the coefficient of variation (CV) of Ct values obtained for each copy number/reaction in three different assays performed on 3 different days.

2.7. Determination of parasite load in lamb feces by qPCR

The developed qPCR method and McMaster counting method were used to monitor the Eimeria spp. infection quantity of four naturally infected lambs for 4 weeks and to compare the difference between two methods.

2.8. Statistical analysis

Data are presented as mean ± S.D. The pMD18T-168 DNA levels are presented as a log unit. GraphPad Prism (www.graphpad-prism.cn) was used for statistical analysis.

3.1 PCR amplification of 18S rRNA region gene of Eimeria spp.

The PCR amplification product of Eimeria spp. 18S rRNA was detected by 1% agarose gel electrophoresis, and the amplified fragment was between 100 bp and 250 bp (Fig. 1), which was basically consistent with the expected fragment (168 bp).

3.2 Identification of recombinant plasmid

The sequencing results of positive clones were compared with sequences (MW512853.1) in GenBank, with a matching rate of 100% (Fig. 2).

3.3 Development of qPCR for Eimeria spp.

The qPCR was performed using different copy numbers of the pMD18T-168 plasmid. The linear dynamic range of the assay was 6 logs (Fig. 3). Assay linearity was established (R² = 0.99779) between dilutions containing 3.43×10³–10⁸ copies/reaction. The standard curve slope was − 3.17, which resulted in a high amplification efficiency of 107%. The results confirmed that, over a wide concentration range, the method had a good linearity and can be used for the detection of target DNA.

3.4 Standardization of qPCR for Eimeria spp.

The specificity of the qPCR was confirmed because no fluorogenic signal was detected when gDNA of G. duodenalis, Blastocystis, Microsporidia, or C. parvum were used (Fig. 4). The limit of detection of qPCR was 34 copies/reaction (Table 1), enabling it to detect samples containing a single oocyst of Eimeria spp. (Table 2). The CV of repeatability tests (intra- and interassay) was less than 2%, indicating that the method had good repeatability (Table 3).

Table 1

Determination of limit of detection for the *Eimeria* spp. qPCR
Plasmid concentration (copies/µL)	Mean Ct value (n = 3)
3.43×10⁸	11.87
3.43×10⁷	17.04
3.43×10⁶	20.76
3.43×10⁵	25.11
3.43×10⁴	28.1
3.43×10³	30.22
3.43×10²	30.73
3.43×10¹	31.87
3.43×10⁰	-
Ct, cycle threshold.
-, No Ct value.

Table 2

Determination of gDNA of oocyst(s) by the *Eimeria* spp. qPCR
Oocyst density	Mean Ct value (n = 3)
10 000	15.95
1000	20.6
100	24.47
10	29.64
1	32.76
Ct, cycle threshold.

Table 3

Intra- and interassay reproducibility tests of qPCR
Plasmid concentration (copies/µL)	Intraassay			Interassay
Plasmid concentration (copies/µL)	Mean Ct value	S.D.	CV	Mean Ct value	S.D.	CV
3.43*10⁸	11.68	0.22	1.88%	11.77	0.10	0.81%
3.43*10⁷	17.40	0.12	0.69%	17.14	0.23	1.33%
3.43*10⁶	20.62	0.24	1.16%	20.82	0.23	1.11%
3.43*10⁵	24.82	0.29	1.16%	24.96	0.15	0.58%
3.43*10⁴	27.24	0.32	1.17%	27.82	0.50	1.80%
Ct, cycle threshold.
S.D., standard deviation.

3.5. Determination of parasite load in lamb feces by qPCR

Figure 5 shows the results of the comparison of the established fluorescence-based quantitative method and McMaster counting method used to measure the infection intensity of four lambs naturally infected with coccidia for 4 consecutive weeks. According to the results, the trend in changes of oocyst infection intensity in the four lambs detected by the two methods was roughly consistent, indicating that the fluorescence-based quantitative method can be used for quantitative detection of coccidia infection in sheep.

In the present study, the 18S rRNA site of the DNA from several species of Eimeria spp. derived from sheep and goats was used to design primers, because this is a highly conserved region among species (Ogedengbe et al. 2011).

A standardized qPCR is expected to provide sensitive, accurate, and reproducible results. One essential requirement for developing a standardized PCR test is the availability of a common standard that is easy to produce in large amounts, is stable, and shows consistent quality across different production batches (Berizi et al. 2021). Recombinant plasmids are stable and easy to standardize, and are often used as standard materials to construct qPCR standard curves (Nunes et al. 2019; Xu et al. 2016). In this study, a plasmid DNA of Eimeria spp., pMD18T-168, was constructed and used as a calibrator to develop a reliable absolute quantitative qPCR for quantifying parasite load in biological samples.

The results of tests revealed that the assay could reliably detect 34 copies of the cloned Eimeria amplicon per µL of fecal DNA extract, and it was possible to find samples containing a single oocyst of Eimeria spp. This demonstrated that the assay showed high sensitivity and could be a useful tool for routine diagnosis. The results resemble those obtained by Kokuzawa et al., who used a conventional PCR, which was also based on the 18S rRNA site, to amplify a single oocyst of this protozoan, with the goal of subsequent sequencing and evaluation of genetic variability among oocysts from the same species (Kokuzawa et al. 2013). The sensitivity of the method established in this study is more sensitive than the qPCR established to diagnose Eimeria spp. in sheep, with a sensitivity of 80 copies per µL of fecal DNA extract reported by Yang et al. (2014). In addition, the established method is also suitable for goats, because the primers were designed from the 18S rRNA gene sequences of Eimeria spp. of both sheep and goats.

Fluorescence-based quantitative PCR is widely used in parasite detection. For example, RT-PCR was used to quantitatively detect the amount of Cryptosporidium oocysts in mouse fecal samples to study the ovulation rule of immunosuppressed and non-immunosuppressed mice infected with Cryptosporidium mouse (Huang et al. 2021). A quantitative RT-PCR was established to study the growth inhibition effect of drugs on Cryptosporidium parvum (Zhang and Zhu 2020). In this study, the developed qPCR method was used to detect the intensity of coccidia infection in lambs naturally infected with Eimeria, and the results were similar to those of routine detection, indicating that the established qPCR can be used to detect the intensity of intestinal coccidia infection in lambs.

A plasmid DNA of Eimeria spp., pMD18T-168, was constructed and used as a calibrator to develop a reliable qPCR for quantifying parasite load in biological samples. The method was shown to be highly sensitive, accurate, and reproducible. The qPCR method was successfully applied for quantifying parasite load of Eimeria spp in lamb feces. The method could be used to evaluate the effect of anticoccidial drugs and growth and reproduction of Coccidia in vitro and in vivo.

Funding This work was financed supported by China Agriculture Research System of MOF and MARA (CARS-38); and Ningxia Hui Autonomous Region Yellow River Basin Ecological Environment Protection and High-quality Development Science and Technology Support Project (No.2021BEF02026).

Conflict of interests The authors declare no competing interests.

Data availability All data generated or analyzed during this study are included in this published article.

Author contributions Conceptualization: Shijie Li and Yichen Jian; Methodology: Shijie Li and Fuchun Jian; Formal analysis: Shijie Li and Kaihui Zhang; Writing - original draft preparation: Shijie Li and Fuchun Jian; Writing - review and editing: Fuchun Jian and Xiaoying Li; Funding acquisition: Fuchun Jian; Supervision: Rongjun Wang and Longxian Zhang. All authors reviewed the manuscript.

Ethics approval All the procedures were conducted according to the Animal Ethics Procedures and Guidelines of the People’s Republic of China.

Consent to participate Not applicable.

Consent for publication All authors consent to be published.

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Development of a qPCR to diagnose the genus Eimeria in sheep and goats

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Abstract

Figures

1. Introduction

2. Materials And Methods

2.1. Samples

2.2. Primer design

2.3. DNA extraction

2.4. Preparation of plasmid standard

2.5. Construction of standard curves for plasmid copy number determination

2.6. Standardization of the qPCR

2.7. Determination of parasite load in lamb feces by qPCR

2.8. Statistical analysis

3. Results

3.1 PCR amplification of 18S rRNA region gene of Eimeria spp.

3.2 Identification of recombinant plasmid

3.3 Development of qPCR for Eimeria spp.

3.4 Standardization of qPCR for Eimeria spp.

3.5. Determination of parasite load in lamb feces by qPCR

4. Discussion

5. Conclusion

Declarations

References

Additional Declarations

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