Aboelsoued Dina, Toaleb Nagwa I, Abdel Megeed Kadria N
Parasitology and Animal Diseases Department, Veterinary Research Institute, National Research Centre, El Buhouth St., Dokki, Giza, Egypt.
AMB Express. 2025 Jan 22;15(1):12. doi: 10.1186/s13568-024-01817-x.
Cryptosporidium sp. is an obligatory intracellular apicomplexan protozoan parasite that causes a disease called cryptosporidiosis with substantial veterinary and medical importance. Therefore, this study aimed to evaluate an early diagnosis of cryptosporidiosis using the anti-Cryptosporidium parvum oocyst immunoglobulin IgG polyclonal antibodies (anti-C. parvum IgG PAbs)-based sandwich enzyme-linked immunosorbent assay (ELISA) for the detection of Cryptosporidium oocyst antigens in fecal samples of farm animals in Egypt. Further molecular identification and sequencing were performed for the detected isolates. Eight hundred and twenty fecal samples of farm animals; 102 buffalo calves, 120 cattle calves, 100 lambs and 98 goat kids, 80 buffaloes, 60 cattle, 160 sheep and 100 goats, collected from different small-scale farms and local holders were examined for cryptosporidiosis by Modified Ziehl-Neelsen (MZN) technique. The percentage of positivity was 45.1%, 50%, 20%, 18.4%, 31.25%, 38.3%, 18.8%, and 11% in buffalo calves, cattle calves, lambs, goat kids, adult buffaloes, adult cattle, sheep, and goats, respectively. Molecular identification of Cryptosporidium samples was performed based on COWP gene, revealing the isolates: GenBank: OQ121955.1, OR029973.1 and PP316107.1 which were identical to the C. parvum and GenBank: PP316108.1 and OR029972.1 which were identical to C. hominis and C. andersoni, respectively. Then, C. parvum oocysts were used for preparation of antigens and rabbit immunization. Anti-C. parvum IgG PAbs were purified and characterized by SDS-PAGE and then labeled with horseradish peroxidase (HRP). Anti-C. parvum IgG PAbs in-house sandwich ELISA was prepared, then tested this ELISA on 820 samples and compared results with MZN microscopical examination and a commercial sandwich ELISA kit. In this study, in-house sandwich ELISA scored higher sensitivity of 98%, 100% specificity, validity 99% and relative agreement 98.6% than (92%, 90%, 91% and 91.4%) of MZN and (96%, 95%, 95.5% and 95.7%) of coproantigen commercial sandwich ELISA kit, respectively. Moreover, we used PCR to evaluate the positivity of in-house sandwich ELISA results, and the total PCR positive samples were 263 out of 268 sandwich ELISA positive samples (98.13%). In conclusion, the prepared Anti-C. parvum IgG PAbs based sandwich ELISA offered a simple and accurate diagnostic method for cryptosporidiosis in the fecal samples of different species of farm animals in Egypt with high sensitivity (98%) and specificity (100%). Further studies on this Anti-C. parvum IgG PAbs may help also in the protection against cryptosporidiosis.
隐孢子虫属是一种专性细胞内顶复门原生动物寄生虫，可引发一种名为隐孢子虫病的疾病，具有重大的兽医和医学意义。因此，本研究旨在评估基于抗微小隐孢子虫卵囊免疫球蛋白IgG多克隆抗体（抗微小隐孢子虫IgG多抗）的夹心酶联免疫吸附测定（ELISA）对埃及农场动物粪便样本中隐孢子虫卵囊抗原的检测，以实现隐孢子虫病的早期诊断。对检测到的分离株进行了进一步的分子鉴定和测序。从不同的小型农场和当地养殖户收集了820份农场动物粪便样本，包括102头水牛犊、120头牛犊、100只羔羊、98只山羊羔、80头水牛、60头牛、160只绵羊和100只山羊，采用改良齐-尼氏（MZN）技术检测隐孢子虫病。水牛犊、牛犊、羔羊、山羊羔、成年水牛、成年牛、绵羊和山羊的阳性率分别为45.1%、50%、20%、18.4%、31.25%、38.3%、18.8%和11%。基于COWP基因对隐孢子虫样本进行分子鉴定，结果显示分离株：GenBank: OQ121955.1、OR029973.1和PP316107.1与微小隐孢子虫相同，GenBank: PP316108.1和OR029972.1分别与微小隐孢子虫和安氏隐孢子虫相同。然后，使用微小隐孢子虫卵囊制备抗原并免疫兔子。纯化抗微小隐孢子虫IgG多抗，并通过SDS-PAGE进行表征，然后用辣根过氧化物酶（HRP）标记。制备了抗微小隐孢子虫IgG多抗内部夹心ELISA，然后在820份样本上测试该ELISA，并将结果与MZN显微镜检查和商用夹心ELISA试剂盒进行比较。在本研究中，内部夹心ELISA的灵敏度为98%，特异性为100%，有效性为99%，相对一致性为98.6%，分别高于MZN的（92%、90%、91%和91.4%）和粪抗原商用夹心ELISA试剂盒的（96%、95%、95.5%和95.7%）。此外，我们使用PCR评估内部夹心ELISA结果的阳性率，在268份夹心ELISA阳性样本中，总PCR阳性样本为263份（98.13%）。总之，所制备的基于抗微小隐孢子虫IgG多抗的夹心ELISA为埃及不同种类农场动物粪便样本中的隐孢子虫病提供了一种简单准确的诊断方法，具有高灵敏度（98%）和特异性（100%）。对这种抗微小隐孢子虫IgG多抗的进一步研究可能也有助于预防隐孢子虫病。
