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Micromanipulation System for Isolating a Single Cryptosporidium Oocyst.

ABSTRACT: In this paper, an integrated system for contact micromanipulation of Cryptosporidium oocysts is presented. The system integrates five actuators and a partially automated control system and contacts the oocyst using a drawn glass end effector with tip dimensions of 1 ? m. The system is intended to allow single cell analysis (SCA) of Cryptosporidium-a very harmful parasite found in water supplies-by isolating the parasite oocyst of 5 ? m diameter in a new environment. By allowing this form of analysis, the source of Cryptosporidium can be found and potential harm to humans can be reduced. The system must overcome the challenges of locating the oocysts and end effector in 3D space and contact adhesion forces between them, which are prominent over inertial forces on this scale. An automated alignment method is presented, using the Prewitt operator to give feedback on the level of focus and this system is tested, demonstrating alignment accuracy of <2 ? m. Moreover, to overcome the challenge of adhesion forces, use of dry and liquid environments are investigated and a strategy is developed to capture the oocyst in the dry environment and release in the liquid environment. An experiment is conducted on the reliability of the system for isolating a Cryptosporidium oocyst from its culture, demonstrating a success rate of 98%.

SUBMITTER: Penny H

PROVIDER: S-EPMC7019727 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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Micromanipulation System for Isolating a Single Cryptosporidium Oocyst.

Penny Hamish H Hayman David T S DTS Avci Ebubekir E

Micromachines 20191218 1

In this paper, an integrated system for contact micromanipulation of Cryptosporidium oocysts is presented. The system integrates five actuators and a partially automated control system and contacts the oocyst using a drawn glass end effector with tip dimensions of 1 μ m. The system is intended to allow single cell analysis (SCA) of Cryptosporidium-a very harmful parasite found in water supplies-by isolating the parasite oocyst of 5 μ m diameter in a new environment. By allowing thi ...[more]

PMID: 31861300

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Project description:Background and aimCryptosporidiosis is a leading cause of diarrheal disease worldwide and is an animal and public health burden. This study aimed to evaluate the protective potential of affinity-purified Cryptosporidium parvum oocyst antigen as a vaccine candidate according to fecal oocyst shedding, humoral and cellular immune responses, histopathological changes, and the number of parasite developmental stages in ileal and hepatic tissues.Materials and methodsWe isolated oocysts from naturally infected buffalo calves and identified them molecularly as C. parvum isolates (GenBank: ON730707 and ON730708) by targeting the Cryptosporidium oocyst wall protein gene. We propagated the C. parvum oocysts in mice. In addition, we prepared crude antigen from the isolated oocysts by purification using cyanogen bromide-activated Sepharose-4B affinity chromatography coupled with rabbit hyperimmune serum. Then, we divided 81 parasite-free mice into three groups: (1) non-vaccinated non-infected mice, (2) mice orally infected with 1 × 105 C. parvum oocysts on week 4 of the experiment, and (3) mice immunized twice with 40 μg/kg of the purified fraction at 2-week intervals. Then, we challenged the vaccinated group with C. parvum oocysts after 2 weeks, and the positive control group was infected at the same time.ResultsWe observed a prolonged prepatent period and decreased oocyst shedding in the vaccinated infected mice compared with the non-vaccinated infected mice (t < 0.001). The vaccinated mice had significantly higher immunoglobulin G levels than those in the other two groups at all examined weeks. In addition, the production of cytokines interferon-gamma, interleukin (IL)-10, IL-12, and IL-15 was activated post-vaccination. After the challenge, all tested cytokines were significantly increased (p < 0.001) in the two infected groups compared with the non-vaccinated non-infected group, with the highest levels in the vaccinated infected group. Vaccinated infected mice exhibited significantly fewer pathological lesions in the ileum and liver than non-vaccinated infected mice, which showed prominent histopathological lesions. Endogenous developmental stages of C. parvum indicated that the ileum was more parasitized than the liver and that vaccination resulted in a lower number of oocysts in ileal and hepatic tissues (p < 0.05).ConclusionOur prepared affinity-purified vaccine candidate could be promising in protecting against cryptosporidiosis.

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Micromanipulation System for Isolating a Single Cryptosporidium Oocyst.

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Micromanipulation System for Isolating a Single <i>Cryptosporidium</i> Oocyst.

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