In vitro method for quantifying and assessing the viability of oocysts of eimeria spp

Abstract

This document belongs to the field of biology and veterinary medicine, presenting a new and simplified in vitro method for differentiating, quantifying and assessing the viability of Eimeria spp oocysts using an iodine-based reagent.

Description

"In vitro method for quantification and evaluation of the viability of Eímería spp. oocysts." FIELD OF INVENTION

[0001] This document belongs to the field of Biology and Veterinary Medicine, presenting a new and simplified in vitro method of differentiation, quantification and viability assessment of Eímería spp oocysts using an iodine-based reagent. DESCRIPTION OF THE STATE OF THE TECHNIQUE

[0002] Eimeriosis, or coccidiosis, is a common animal disease caused by protozoa of the genus Eímeria, particularly in poultry. It is an infection that usually affects young animals and can lead to the death of entire poultry farms, directly impacting the economy of rural producers.

[0003] Therefore, the main current strategy for preventing coccidiosis in these animals is through flock vaccination. Coccidiosis vaccines are produced from the oocysts of the parasite Eímería spp.

[0004] Currently, the viability analysis of Eímería oocysts is done by inoculating the oocyst suspension into birds and waiting for the incubation time of the oocysts in the birds in order to verify the pathological effects caused by the inoculated oocysts.

[0005] When the oocysts are not viable, the birds do not show any degree of intensity of the effects pathological characteristics of the oocysts compared to non-inoculated birds. This technique is costly due to the need for a dedicated animal handling area to obtain results after days, and it also presents a challenge because it involves testing on in vivo animals.

[0006] Furthermore, the viability assessment method currently used in the market presents qualitative results, and it is not possible to quantify the viability of oocysts in percentage terms for their subsequent application in obtaining APIs for the production of vaccines to prevent coccidiosis, a very common disease in birds that can lead to significant losses in industrial poultry farming. PRIORITY OF THE INVENTION

[0007] US Patent 6,344,340 BI discloses a viability assay for sporocyst-forming protozoa, in which a sample of the protozoan is treated with at least one vital dye in order to determine the viability of the protozoan based on color differentiation using fluorescence microscopy. Therefore, it completely diverges from the method proposed by the subject of investigation, which proposes a colorimetric method using an iodine-based reagent to identify viable Eímeria oocysts in a more simplified manner, using a counting chamber in an optical microscope.

[0008] Patent document IL176475A discloses a method for purifying Eímeíra spp. oocysts from fecal material using enzymes. The method consists of placing the fecal material in contact with polysaccharidase, such as cellulase and pectinase, and subsequently amylase, in order to remove such material. Therefore, it completely diverges from the method proposed by the claimed invention, which proposes a colorimetric method using an iodine-based reagent to identify viable Eimeria oocysts, as well as their differentiation from other particles, such as starch.

[0009] Patent document AU200031298A discloses a method for identifying oocysts involving antibody binding, in addition to using another species of protozoan (Cryptosporidium spp.). Therefore, it completely diverges from the method proposed by the present invention, which proposes a colorimetric method using an iodine-based reagent for identifying viable Eimeria oocysts.

[0010] Patent US7229615B2 discloses a method for producing vaccines, part of the claims of which relate to the sporulation and separation of Eimeria oocysts for the production of such vaccines. More specifically, the described method utilizes water and hydrogen peroxide, followed by aeration of the material and pH conditioning to recover the oocysts from the material. Therefore, it differs completely from the method proposed by the present invention, which proposes a colorimetric method using an iodine-based reagent to identify viable Eimeria oocysts.

[0011] Patent document CN113367099A discloses the use of iodine solution in eggs in a method for obtaining coccidiosis-free birds for use in the field. experimental. Therefore, it completely diverges from the method proposed by the research object, which proposes a colorimetric method using an iodine-based reagent to identify viable Eimeria oocysts.

[0012] The article "A simple and efficient method for isolation of a single Eimeria oocyst from poultry litter using a micromanipulator" (Dongjean Yim et al., 2011) reveals the use of a micromanipulator, as well as genomic identification, to isolate a single Eimeria oocyst from chicken fecal material. Therefore, it completely diverges from the method proposed by the present invention, which proposes a colorimetric method using an iodine-based reagent to identify viable Eimeria oocysts.

[0013] The article "Single oocyst infection: a simple method for isolation of Eimeria spp. from the mixed field samples" (R.E. Khalafalla et al., 2010) reveals two simple methods for morphological identification of sporulated oocysts of isolated Eimeria spp. The first describes the use of gelatin, and the second describes a DNA extraction method for oocyst identification. Therefore, it completely diverges from the method proposed by the research subject, which proposes a colorimetric method using an iodine-based reagent for identification of viable Eimeria oocysts.

[0014] Therefore, the use of an iodine-based solution for differentiation, quantification and assessment of oocyst viability is not described in the state of the art. viable Eimeria spp. in a simplified and fully in vitro method. SUMMARY OF THE INVENTION

[0015] The present invention is presented and characterized in the independent claims, while the dependent claims describe other features of the invention or embodiments related to the main inventive idea.

[0016] In a first aspect, the present invention claims the use of an iodine-based solution in the quantification and in vitro evaluation of the viability of Eimeria spp. oocysts, using a counting chamber in an optical microscope.

[0017] In a second aspect, the invention claims that the iodine solution used has as a formulation 0.4% to 3% resublimated iodine; 1.8% to 5% potassium iodide; 70% to 85% alcohol; 25% to 35% purified water.

[0018] In a third aspect, the invention claims that oocyst differentiation occurs through the differentiation of stained and unstained particles, with viable oocysts becoming translucent, while starch particles and / or non-viable oocysts become stained. Additionally, the present invention provides that the counting can be performed manually and / or using an image analysis system.

[0019] In a fourth aspect, the invention claims that The counting chambers used in optical microscopes can be Neubouer, Fuchs Rosenthal, Petrof f-Hausser, Sedgwick-Rafter or Burker chambers.

[0020] In a fifth aspect, the present invention claims that the methodology can be applied in verifying the condition of oocyst bulks, in defining the stability time of Active Pharmaceutical Ingredients and vaccines, in quality control of the manufacturing process of vaccines against coccidiosis, in evaluating improvements in the production process, in estimating the level of vaccine response of the vaccine against coccidiosis in vitro. OBJECTIVES OF THE INVENTION

[0021] The main objective of this invention is to propose an innovative and simplified technique for the quantitative evaluation of viable oocysts of Eimeria spp., in addition to their differentiation from other particles and non-oocysts (such as starch, for example) by staining oocysts in suspension using an iodine-based reagent.

[0022] Another objective of the present invention is to optimize the process of evaluating the viability of oocysts, without the need for testing on animals, as this method can be performed in vitro.

[0023] Another objective of the present invention is to facilitate the counting of oocysts with the presence of a high amount of starch in the suspensions and the reduction of analytical errors in the differentiation of oocysts from non-oocysts, in addition to facilitating the quantification of the suspensions. BRIEF DESCRIPTION OF THE FIGURES

[0024] The characteristics, aspects and advantages of the present invention will be better understood in relation to the following description and its claims from the attached figures, in which:

[0025] Figures 1 and 2 demonstrate the visualization of the difference between stained and unstained oocysts under an optical microscope.

[0026] Figures 3 and 4 demonstrate conventional oocyst quantification.

[0027] Figures 5 and 6 demonstrate the quantification of oocysts using the iodine solution. DETAILED DESCRIPTION OF THE INVENTION

[0028] This description aims to further explain the inventive concept, provide examples to facilitate its understanding, and provide precise technical data on some of the ways to implement the inventive concept of the invention. The detailed description also aims to prevent third parties from repeating the extensive experimentation, financial investments, time, and intellectual activity that the inventors / applicant made to solve the technical problems herein solved.

[0029] Oocyst counting is currently performed using the Neubour chamber for process control and the formulation of coccidiosis vaccines. The vaccine manufacturing process is based on the collection of feces from birds infected with the oocysts.

[0030] Due to the matrix in which oocysts are found (bird feces), several particles are found that significantly affect oocyst identification, the main presence being starch. To improve oocyst identification from these particles, and to further differentiate viable from nonviable oocysts, a reagent containing iodine was added.

[0031] Iodine binds to starch molecules, causing their dark coloration, facilitating the differentiation of starch particles, which remain dark, from oocysts, which remain translucent.

[0032] With this technique, it is also possible to observe that some oocysts are stained with a less intense color than starch. It is also observed that these stained oocysts present deformations characteristic of non-viable oocysts.

[0033] After several experiments and sample analyses, it was observed that other oocysts, despite appearing normal, were being stained by the reagent. Furthermore, by performing a forced oocyst inactivation procedure, it was possible to determine the correlation between the percentage of viability loss and the stained oocysts. Thus, it was possible to determine that the claimed method is useful for differentiating starch oocysts and also for assessing oocyst viability.

[0034] The iodine solution used for the methods claimed by the present invention has the following formulation:

[0035] - 0.4% to 3% resublimated iodine;

[0036] - 1.8% to 5% potassium iodide;

[0037] - 70% to 85% alcohol;

[0038] - 25% to 35% purified water.

[0039] The first method using the aforementioned iodine solution claimed in the present invention is carried out by a simple procedure, the steps of which are described below:

[0040] a. Dilute the iodine solution in distilled water in a ratio of 1:0.2 to 1:20 at a temperature of 15-30°C at the time of use;

[0041] b. Aliquot the diluted iodine solution into tubes;

[0042] c. Keep the Eímería suspension being analyzed in the temperature range between 15°C and 30°C;

[0043] d. Homogenize the Eímería suspension and transfer it to the tube containing the Iodine solution from a);

[0044] e. Optionally dilute the Eímería suspension in serial dilutions using the iodine solution when necessary;

[0045] f. Wait for up to 35 minutes;

[0046] g. Quantify the difference in stained and unstained oocysts in a light microscope counting chamber, such as a Neubauer chamber.

[0047] The second method claimed in the present invention is carried out by the steps described below:

[0048] i) Dilute the Iodine Solution between 1:0.1 and 1:10 in distilled water refrigerated between 2 and 8 °C at the time of use;

[0049] ii) Aliquot the diluted iodine solution into bottles suitable for serial dilution;

[0050] iii) Keep the Eímería suspension to be analyzed refrigerated in a temperature range between 2 and 8 °C;

[0051] iv) Homogenize the Eímería suspension;

[0052] v) Transfer the suspension to the vial containing the iodine solution (1:1) and then proceed with serial dilution using the iodine solution until the ideal dilution for the study is reached;

[0053] vi) Quantify the number of Eímería spp. oocysts found in an optical microscope chamber.

[0054] The method described above allows the differentiation of Eímería spp oocysts from non-oocysts by facilitating the counting of oocysts with the presence of a high amount of starch in the suspensions, enabling improvements in the counting process and reducing analytical errors in differentiation.

[0055] Both of the above methods provide quantitative information that can be useful in the following situations:

[0056] Check the condition of oocyst bulks (IFAs) before their formulation, adjusting the formula according to the % viability, allowing the production of vaccines that have the same quality levels in different batches produced.

[0057] Define the stability time of IFAs and vaccines according to the temperature and storage conditions.

[0058] Check the storage of IFAs and vaccines produced (which are negatively affected by storage temperature), whether they were at the ideal temperature or whether they were exposed to temperatures outside the specification.

[0059] Manufacturing process control of coccidiosis vaccines, used to assess the loss of viability during the production process, in order to assist in validating the decontamination of surfaces, materials and processes.

[0060] Evaluate production process improvements due to the inclusion of solutions to reduce loss of viability.

[0061] Estimate the level of the vaccine's vaccinal response without the need for animal testing.

Claims

MODIFIED CLAIMS Received by the International Secretariat on 6 January 2025 (06.01.2025) 1. In vitro method for evaluating the viability of Eimeria spp. oocysts, characterized by comprising the steps of a) Sampling a volume of oocyst suspension Eimeria spp. and mix with a volume of iodine-based solution, b) Add the mixture obtained in a) to a counting chamber, c) Count stained and unstained Eimeria spp. oocysts using an optical microscope to determine oocyst viability.

2. In vitro method for quantification of oocysts of Eimeria spp. characterized by understanding the stages of D Dilute the iodine solution in water, ii) Aliquot the diluted iodine solution into containers suitable for serial dilution, iii) Homogenize a suspension of Eimeria oocysts, iv) Transfer the Eimeria suspension to the container with the iodine solution at a 1:1 ratio and continue with serial dilution using the iodine-based solution until the desired dilution is reached, v) Add the diluted solution from iv) to a counting chamber, vi) Quantify the number of Eimeria spp. oocysts under an optical microscope.

3. Method according to claim 1, characterized in that the proportion between the sample and the iodine-based solution from step a) is between 1:0.2 and 1:

20.

4. Method according to claim 2, characterized in that the ratio between the iodine-based solution and water from step i) is between 1:0.1 and 1:10, and the temperature is between 2-8°C at the time of use.

5. Method according to any one of the preceding claims, characterized in that the iodine-based solution has a formulation of 0.4% to 3% resublimated iodine; 1.8% to 5% potassium iodide; 70% to 85% alcohol; 25% to 35% purified water.

6. Method according to any one of the preceding claims, characterized in that the differentiation of starch particles and / or non-viable oocysts in relation to viable oocysts is through the dark coloration acquired by the starch particles and / or non-viable oocysts when treated with the iodine-based solution as described in claim 5.

7. Method according to any one of the preceding claims characterized by counting oocysts of Stained and unstained Eimeria spp. and / or the quantification of the number of Eimeria spp. can be performed manually and / or using an image analysis system.

8. Method according to any one of the preceding claims, characterized in that the counting chamber in an optical microscope is, optionally, any one of Neubauer chamber, Fuchs Rosenthal chamber, Petrof f -Haus ser , Sedgwick-Raf ter chamber or chamber of Burker.

9. Method according to any one of the preceding claims, characterized in that it is used in checking the condition of oocyst bulks, defining the time of stability of Active Pharmaceutical Ingredients and vaccines, quality control of the manufacturing process of vaccines against coccidiosis, evaluation of improvements in the production process, estimation of the level of vaccine response of the vaccine against coccidiosis in vitro.

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