Methods for species-specific quantitation of eimeria oocysts in mixed vaccine samples

Abstract

The disclosure provides a hybrid protocol for identifying and quantitating viable Eimeria oocysts, such as in a mixed industrial scale vaccine sample. The hybrid protocol comprises the combination of a volumetric viability assessment, which allows for quantifying the number of viable oocysts, and a digital PCR (dPCR) assay, which allows for quantitative determination of the species composition of a mixed sample.

Description

[0001] Docket No.: HVJ-009PC METHODS FOR SPECIES-SPECIFIC QUANTITATION OF EIMERIA OOCYSTS IN MIXED VACCINE SAMPLES Related Applications claims priority to U.S. Provisional Application No. 63 / 657,479, filed June 7, 2024, of which is hereby incorporated by reference. BACKGROUND Coccidiosis is a disease of various animals in which the intestinal mucosa is invaded and damaged by a protozoa of the subclass Coccidia. The economic effects of coccidiosis can be especially severe in the poultry industry where intensive housing of birds favors the spread of the disease. Infection by coccidial protozoa is, for the most part, species specific. Numerous species, however, can infect a single host. For example, there are seven species of coccidial protozoa that infect chickens, six of which are considered to be moderately to severely pathogenic. Two methods are currently used to control coccidiosis in poultry, chemotherapy or vaccination. Numerous drugs are available for the control of coccidiosis in poultry. However, because of the number of species that cause the disease, very few drugs are efficacious against all species, although a single drug may be efficacious against several species. In modern broiler chicken production, for example, administration of drugs to control coccidiosis is routine. The expense for preventative medication against coccidiosis represents a significant cost of production. Vaccination of birds against coccidiosis is an alternative to chemotherapy. An advantage of vaccination is that it can greatly reduce or eliminate the need to administer anti-coccidial drugs, thus reducing drug costs to poultry producers, preventing the development of drug-resistant strains, reducing feed and medicinal mixing holding / mixing tanks, and lessening consumer concerns about drug residues. Numerous methods have been developed to immunize poultry against Eimeria species.. The most effective methods have been based on the administration of controlled dosing of live sporulated oocysts (sporocysts), using either fully virulent strains or attenuated strains. Typically vaccines contain multiple different species of coccidial protozoa. Thus, in both the preparation and use of such vaccines, the ability to identify and quantify the coccidial protozoa, Docket No.: HVJ-009PC particularly live oocysts, is highly advantageous. Early methods to quantify live oocysts was done by inoculation of control birds with a test vaccine, challenging them to see if they were protected, and then calibrating the final vaccine product based on the results from the challenged birds. This approach however, is very time-consuming (e.g., 5 weeks), adds labor to the production cost, requires a licensed animal facility associated with the manufacturing site for vaccine titration, and requires a statistically valid number of animals to determine the appropriate number of live oocysts in each vaccine lot. More recent approaches have been developed that utilized in vitro methods to evaluate viability of live oocysts, which include staining methods to identify live oocysts or sporocysts (see e.g., PCT Publication WO 2012 / 122561 and Agullo-Barcelo et al. (2014) Lett. Appl. Microbiol. 58- 70-78) and quantitative DNA approaches (see e.g., PCT Publication WO 2011 / 043737; European Application EP0516381, Japanese Application JP2007020543; and Nolan et al. (2015) Parasitol. Intl. 64:464-470, US patent 6,344,340). While certain advances have been made, there is still a need in the art for methods to accurately identify and quantify Eimeria protozoa in mixed vaccine samples in large scale vaccine production containing 106to 1010oocysts, specifically as it applies to optimizing production / sampling methods and determining the number of live oocysts in a finished and filled vaccine serial. SUMMARY The disclosure provides methods of identifying and quantifying Eimeria species, such as in mixed vaccine samples, that can be applied effectively on an industrial scale. The methods of the disclosure use a hybrid approach for identifying and quantifying Eimeria oocysts that comprises the combination of a volumetric viability assessment, which allows for quantifying the number of viable oocysts, and a digital PCR (dPCR) assay, which allows for quantitative and identity determination of the species composition of a mixed sample. Accordingly, in one aspect, the disclosure pertains to a method for determining the number and proportion of viable Eimeria oocysts relative to non-viable Eimeria oocysts in a sample comprising a plurality of Eimeria species, the method comprising: determining the number of viable Eimeria oocysts relative to non-viable Eimeria oocysts in the sample; Docket No.: HVJ-009PC extracting DNA from the viable Eimeria oocysts; subjecting the extracted DNA to digital PCR (dPCR) reactions producing amplicons specific for each Eimeria species in the sample; and determining the proportion of viable Eimeria oocysts for each Eimeria species in the sample. In embodiments, the number of viable Eimeria oocysts relative to non-viable Eimeria oocysts is determined by staining the viable and non-viable oocysts with a vital dye, such as a fluorescent dye, a nucleic acid staining dye, or a propidium derivative. In embodiments, the vital dye is propidium monoazide. In embodiments, the staining is carried out in the presence of one or more non-ionic surfactants, non-limiting examples of which include Triton X-100, Tween 80, or both. In embodiments, the number and / or proportion of viable oocysts is based on the extent of staining by the non-viable oocysts. In embodiments, the extent of staining is determined by fluorescence microscopy. In embodiments, the extent of staining is determined by flow cytometry. In embodiments, the step of extracting the DNA is carried out following isolation of the viable oocysts by fluorescence-activated cell sorting (FACS). In embodiments, the method of the disclosure comprises: (a) staining oocysts from a vaccine preparation comprising one or more mixed population of sporocyst-forming Eimeria species with a vital dye; (b) determining the number and proportion of viable oocysts for each of species based on the extent of staining in step (a); (c) isolating the viable oocysts from the non-viable oocysts; (d) excysting sporocysts from the viable oocysts and extracting DNA from the sporocysts; (e) subjecting the DNA extracted in step (d) to species-specific digital PCR (dPCR) reactions comprising sequenced characterized amplified region (SCAR) primers specific for each of the one or more sporocyst-forming protozoa species; (f) synthesizing species-specific SCAR amplicons from the dPCR reactions; (g) determining the copy number of each species-specific SCAR amplicon produced in step (f); and Docket No.: HVJ-009PC (h) determining the number and proportion of sporulated oocysts relative to unsporulated oocysts for each of the one or more sporocyst-forming species in the mixed population, based on the results in steps (b) and (g). In embodiments, the sample comprises a plurality of different sporocyst-forming protozoa species. In embodiments, the SCAR primers comprise two or more nucleotide sequences selected from the group consisting of SEQ ID NOs: 1-9. In other embodiments, the SCAR primers comprise two or more nucleotide sequences selected from the group consisting of SEQ ID NOs: 10-30. In embodiments, the copy number of each species-specific SCAR amplicon is determined by comparison to a standard curve generated from known amounts of polynucleotides corresponding to each of the species-specific amplicons. In embodiments, the dPCR assays are corrected for DNA extraction efficiency, PCR efficiency and / or SCAR amplicon copy number, wherein the mixed sample and the SCAR pDNA for each species are subjected to a SCAR dPCR assay, and wherein correction factors for each assay are determined to calculate the original concentrations of live sporulated oocysts per species. In embodiments, the vital dye is a fluorescent dye, a nucleic acid staining dye, or a propidium derivative. In embodiments, the vital dye is propidium monoazide. In embodiments, the staining is carried out in the presence of one or more non-ionic surfactants, such as Triton X-100, Tween 80, or both. In embodiments, the staining in step (a) is carried out in the presence of a brief heat treatment. In embodiments, the extent of staining is determined by the number of non-viable oocysts. In embodiments, the extent of staining is determined by fluorescence microscopy. In embodiments, the extent of staining is determined by flow cytometry. In embodiments, the lower limit of detection (LoD) for viable oocysts is 1-5 oocysts per species. In embodiments, the oocyst sample comprises oocysts from least two Eimeria species selected from the group consisting of E. mitis, E. acervulina, E. tenella, E. maxima, E. necatrix, E. praecox, and E. brunetti. In embodiments, the oocyst sample comprises oocysts from E. mitis, E. acervulina, E. tenella, and E. maxima. In embodiments, the oocyst sample comprises oocysts from at least two Docket No.: HVJ-009PC Eimeria species selected from the group consisting of E. 29innocua, and E. subrotunda. In embodiments, the oocyst sample comprises oocysts from E. adenoeides and E. meleagrimitis. In embodiments, the sample is a fecal sample. In another aspect, the disclosure pertains to a method for determining the viability of Eimeria oocyst samples relative to non-viable oocyst samples recovered at different stages of vaccine production comprising: determining the number and proportion of viable oocysts relative to non-viable oocysts in each of a plurality of oocyst samples recovered following a different stage of vaccine production; extracting DNA from sporocysts excysted from the viable oocysts and subjecting the extracted DNA to dPCR reactions producing amplicons specific for each of the protozoa species in the preparation; determining the copy number of PCR products corresponding to each of the one or more species; and determining the number and proportion and number of viable oocysts relative to non-viable oocysts for each species based on the copy number of amplicons corresponding to each species, wherein the vaccine preparation is derived from a purified and sporulated oocyst sample. In embodiments, the different stages of vaccine production are selected from the group consisting of an initial sample collection stage, an oocyst preparation stage, a species blending stage, a packaging stage, and a combination thereof. In embodiments, the amplicons in step (b) are sequence-specific SCAR amplicons produced by using SCAR primers specific for each of the one or more sporocyst-forming protozoa species. In embodiments, the SCAR primers comprise two or more nucleotide sequences selected from the group consisting of SEQ ID NOs: 1-9. In other embodiments, the SCAR primers comprise two or more nucleotide sequences selected from the group consisting of SEQ ID NOs: 10-30. In embodiments, the copy number of each species-specific SCAR amplicon is determined by comparison to a standard curve generated from known amounts of polynucleotides corresponding to each of the species-specific amplicons. In embodiments, the dPCR assays are corrected for DNA extraction efficiency, PCR efficiency and SCAR amplicon copy number, wherein the mixed sample and the SCAR pDNA for each species Docket No.: HVJ-009PC are subjected to a SCAR dPCR assay, and wherein correction factors for each assay are determined to calculate the original concentrations of live sporulated oocysts per species and the original concentrations of SCAR copies per species in the mixed sample. In embodiments, the vaccine preparation comprises oocysts from at least two species selected from E. mitis, E. acervulina, E. tenella, E. maxima, E. necatrix, E. praecox, and E. brunetti. In embodiments, the vaccine preparation comprises oocysts from E. mitis, E. acervulina, E. tenella, and E. maxima. In embodiments, the vaccine preparation comprises oocysts from at least two species selected from E. meleagridis, E. meleagrimitis, E. gallopavonis, E. adenoeides, E. dispersa, E. innocua, and E. subrotunda. In embodiments, the vaccine preparation comprises oocysts from E. adenoeides and E. meleagrimitis. In embodiments, the method comprises: (a) staining oocyst samples from a vaccine preparation recovered from each of a plurality of different stages of vaccine production, wherein each oocyst sample comprises a plurality of sporocyst- forming protozoa species, and wherein each oocyst sample is stained with a vital dye; (b) determining the number and proportion of viable oocysts relative to non-viable oocysts in each oocyst sample, wherein the number and proportion of viable oocysts relative to non-viable oocysts in each oocyst sample is determined by correlating the extent of staining in each oocyst sample; (c) preparing viable oocyst samples from each oocyst sample in step (b), wherein the viable oocysts in each oocyst sample are separated and isolated away from the non-viable oocysts; (d) excysting sporocysts from each viable oocyst sample in step (c); (e) extracting DNA samples from the excysted sporocysts in step (d); (f) subjecting each DNA sample in step (e) to dPCR reactions comprising a plurality of SCAR primer pairs, wherein each SCAR primer pair is specific for a different sporocyst-forming protozoa species; (g) synthesizing SCAR amplicons produced from each dPCR reaction in step (f); (h) determining the copy number for each SCAR amplicon by comparison to standard curves generated from known amounts of polynucleotides corresponding to each SCAR amplicon; and Docket No.: HVJ-009PC (i) determining the proportion and number of viable oocysts corresponding to each of the protozoa species, based on the results in steps (b) and (h), wherein the vaccine preparation is derived from a fecal sample, and wherein the different stages of vaccine production are selected from the group consisting of an initial sample collection stage, an oocyst preparation stage, a species blending stage, a packaging stage, and a combination thereof. In embodiments, steps (a)-(i) are repeated for a portion of the vaccine preparation tested at different dates relative an expiration date corresponding to the vaccine preparation. In embodiments, the dPCR assays are corrected for DNA extraction efficiency, PCR efficiency and SCAR amplicon copy number, wherein the mixed sample and the SCAR pDNA for each species are subjected to a SCAR dPCR assay, and wherein correction factors for each assay are determined to calculate the original concentrations of live sporulated oocysts per species and the original concentrations of SCAR copies per species in the mixed sample. In embodiments, the vital dye is a fluorescent dye, a nucleic acid staining dye, or a propidium derivative, such as propidium monoazide. In embodiments, the staining is carried out in the presence of one or more non-ionic surfactants, such as Triton X-100, Tween 80, or both. In embodiments, the staining is carried out in the presence of a brief heat treatment. In embodiments, the extent of staining is determined by the number of non-viable oocysts. In embodiments, the number and proportion of viable oocysts is determined by fluorescence microscopy. In embodiments, the number and proportion of viable oocysts in step is determined by flow cytometry. In embodiments, the step of extracting the DNA is carried out following isolation of the viable oocysts by flow cytometry. In embodiments, each oocyst sample comprises species belonging to the Apicomplexa phylum. In embodiments, each oocyst sample comprises species belonging to a genus selected from the group consisting of Eimeria, Isospora, Cryptosporidium, Toxoplasma and combinations thereof. In embodiments, each oocyst sample comprises Eimeria species. In embodiments, each oocyst sample comprises Eimeria species selected from the group consisting of E. mitis, E. acervulina, E. tenella, E. maxima, E. necatrix, E. praecox, and E. brunetti. In embodiments, each oocyst sample comprises a Docket No.: HVJ-009PC mixed population of E. mitis, E. acervuline, E. tenella, and E. maxima. In embodiments, each oocyst sample comprises Eimeria species selected from the group consisting of E. meleagridis, E. meleagrimitis, E. gallopavonis, E. adenoeides, E. dispersa, E. innocua, and E. subrotunda. In embodiments, each oocyst sample comprises a mixed population of E. adenoeides and E. meleagrimitis. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows raw qPCR data and linear regression of monoculture dilution series. FIG. 2 shows SCAR target amplification products for cloning into pCR2.1 vector. FIG. 3 shows pDNA linear regressions with PCR efficiencies for qPCR (upper row) and dPCR (lower row). FIG. 4 shows PMA epifluorescence in the following untreated (blue square) and heat- killed (red square) samples: Panel A, Ega-230119 (MS); Panel B, Ead29May19 (MS); and Panel C, Emt-290119 (MS). FIG. 5 shows PMA staining as observed by FACSMelody: EGA-USUS-28May19 (top row); EAD-USUS-04Aug20 (middle row); and EMT-USUS-21Jun20 (bottom row). FIG. 6 shows linear regressions of SCAR pDNA standards obtained by dPCR. SCAR DNA was transferred to a PCR 2.1 vector for every Eimeria spp. A 10-fold serial dilution was made for each Eimeria spp. sample and 5 µl was subjected to its corresponding SCAR dPCR assay in duplicate. FIG.7 shows correction factors for dPCR assays. Correction factors for dPCR assays with SCAR DNA were obtained by analyzing mixtures of viable E. mitis, E. acervulina and E. tenella that were composed by FACSMelody in different proportions. The differences in the amount of copies per species per gene between what could theoretically be expected and what was actually observed by dPCR resulted in a correction factor (∆Yintercept) when the slope of the mixtures was fixed according to the slope of the pDNA standards for the relevant gene. FIG. 8 shows the volumetric species-specific viability of different vaccine batches according to the hybrid technique in Example 2. MMAT 1000 and 5000 vaccines were subjected to the full hybrid method. For E. maxima volumetric viability was analyzed by flow cytometry. For the Docket No.: HVJ-009PC other species viability was analyzed using a combination of flow cytometry and SCAR dPCR assays. Original concentrations of live sporulated oocysts per species were calculated from OBPR’s. FIGS. 9A-9D show the gating strategy used for MMAT 100 (FIGS. 9A, 9B) and MMAT 5000 vaccines (FIGS.9C, 9D) of different shelf lives. Oocysts were incubated with 0.05% Tween 80 at least overnight. To determine viability oocysts were incubated for 4 minutes at 61°C completed with 6 minutes at RT in the presence of 100 µM PMA, 0.5% Triton X-100 and 0.05% Tween 80 before photoactivation. Samples were analysed by FACS Melody using a 2.0 neutral density filter and an agitation of 300 rpm. Selection of the oocyst population was based on SSC-A and FSC-A and followed by gating based on autofluorescence ((λex:405 nm - λem:528 / 45 nm). PMA-positive and - negative populations were detected using the yellow-green laser and an associated filter set ((λex:: 561 nm - λem: 613 / 18). Viability was determined in triplicate. FIG.10 shows purity of the viable gate. The viable gate was analyzed microscopically for purity and sporulation and this was taken into account in the calculation of the total volumetric viability. FIG.11 shows the number of SCAR copies / µL detected by dPCR before correction. DNA of 35,000 sorted viable oocysts from various MMAT 1000 and 5000 vaccine batches was extracted and subjected to a SCAR dPCR assay. FIG. 12 summarizes the viability of parent and attenuated strains by FACSMelody. Oocysts were incubated for 4 minutes at 61°C completed with 6 minutes at RT in the presence of 100 µM PMA, 0.5% Triton X-100 and 0.05% Tween 80 before photoactivation. Samples were analysed by FACS Melody using a 2.0 neutral density filter and an agitation of 300 rpm. Selection of the oocyst population was based on SSC-A and FSC-A and followed by gating based on autofluorescence (λex:405 nm - λem: 528 / 45 nm). PMA-positive and -negative populations were detected using the yellow- green laser and an associated filter set (λex: 561 nm - λem: 613 / 18). Viability was determined independently in triplicate. FIG. 13 shows the gating strategy used for attenuated and parent strains. Oocysts were incubated with 0.05% Tween 80 at least overnight. To determine viability oocysts were incubated for 4 minutes at 61°C completed with 6 minutes at RT in the presence of 100 µM PMA, 0.5% Triton X- 100 and 0.05% Tween 80 before photoactivation. Samples were analysed by FACS Melody using a Docket No.: HVJ-009PC 2.0 neutral density filter and an agitation of 300 rpm. Selection of the oocyst population was based on SSC-A and FSC-A and followed by gating based on autofluorescence (λex405 nm - λem528 / 45 nm). PMA-positive and -negative populations were detected using the yellow-green laser and an associated filter set (λex: 561 nm - λem: 613 / 18). Viability was determined in triplicate. DETAILED DESCRIPTION The disclosure pertains to a hybrid protocol for identifying and quantifying live Eimeria oocysts, such as in a mixed species vaccine sample. Various aspects of the disclosure are described in further detail in the subsections below. I. Eimeria Species The method of the disclosure can be applied to different Eimeria species, such as Eimeria that infect poultry or cattle. In an embodiment, the method of the disclosure is used with Eimeria species that infect chickens. Examples of Eimeria species that infect chickens include E. maxima, E. acervulina, E. tenella, E. mitis, E. brunetti, E. hagani, E. necatrix and E. praecox. In an embodiment, the methods are used with a mixed vaccine sample comprising E. maxima and at least one, or at least two or at least three other Eimeria species. In an embodiment, the methods are used with a mixed vaccine sample comprising at least E. maxima, E. acervulina and E. tenella. In another embodiment, the method of the disclosure is used with Eimeria species that infect turkeys. Examples of Eimeria species that infect turkeys include E. gallopavonis, E. adenoides and E. meleagrimitis. II. Vaccine Preparation Methods for preparing mixed species Eimeria vaccines are well-established in the art. Various aspects of the methods for large scale isolation, sporulation, purification, and storage of Eimeria oocysts are described by Schasteen, U.S. Pat. No. 7,846,685. The isolation of sporocysts are described by Pfannenstiel, U.S. Pat. No. 6,344,340. Briefly the methods include: Source of oocysts Docket No.: HVJ-009PC Oocysts may be obtained from feces or tissue of infected animals; contaminated feed or water; soil; pen litter or bedding; or a variety of other sources. The exact procedures used will vary with the material from which the oocysts are obtained and will be apparent to those skilled in the art. In one embodiment of the present invention, oocysts are obtained from excreta. Isolation of oocysts Five hundred fifty, 15 day old broiler chickens were infected with approximately 7000 viable oocysts per bird of Eimeria sp. by oral gavage or by ingestion via drinking water or feed. Excreta were collected over a three day period beginning 6 days later at 21 days of age. Total excreta collected over the three day period was 189 kg. Excreta were processed on the day collected. The excreta collected were put in a dilution tank maintained at approximately 40-50° F. and diluted with water at 0.687 to 0.630 L / bird. The diluted excreta was pumped through a 30" diameter vibrating sieve fitted with a 50 mesh (297 micron) top screen and a 250 mesh (61 micron) bottom screen at a rate of approximately 6 LPM. The top two fractions were discarded and the filtrate, containing the oocysts, was pumped into a chilled (about 40-50° F.) collection tank and continuously agitated. The filtrate was then pumped at a feed rate of approximately 2.9-3.5 LPM into a Sharples Super-D-Canter centrifuge. The centrifuge settings were: bowl speed 3990-4004 RPM; auger speed 2306-3990 RPM and RPM delta 16.84-17.33. RPM delta is a measure of the difference between bowl and auger speeds. Total run time ranged from 97 to 100 minutes. The centrate was discarded and the solids (cake), which contained oocysts, were collected into a stainless steel tray, weighed and stored in a tank at about 40- 50° F. The solids obtained from each of three collection days were combined. The volume of solids from the combined three runs was 28 L. To these solids was added 25.6 L of high fructose com syrup and 46.4 L of water to give a total volume of 100 L and a specific gravity of 1.094 g / 1. This material was then centrifuged using a Sharples Super-D-Canter centrifuge at a bowl speed of 5998 RPM, an auger speed of 3998 RPM and a RPM delta of 20.41. The feed rate was 1.1 I / min and the total run time was 95 minutes. The centrate, containing the oocysts, was collected and stored in a tank at approximately 40-50° F. and the unwanted excreta solids discarded. In order to remove the residual sugar in solution and to speed 4050 RPM. The initial RPM delta was 20.01 but was decreased to 15 and then 10 during the run to increase centrate flow. The centrate was discarded and the solid Docket No.: HVJ-009PC containing the oocysts was retained. The oocysts were placed in a sterilized container with sterile water at a preferred concentration of between about 5xl06 / ml and about 50xl06 / ml and held at from about 2° C. to about 8° C. until transferred to the sporulation vessel to undergo sporulation. Sporulation Sporulation was accomplished by adding enough of a 5.25% sodium hypochlorite solution to obtain a final concentration 0.05 wt% sodium hypochlorite. This oocyst / sodium hypochlorite mixture was added to a 10 liter fermentor set at 28+1° C. and an agitation rate of 200 RPM. Oxygen was provided by portable oxygen cylinders and bubbled through the mixture at a rate sufficient to obtain a percent saturation of dissolved oxygen value of at least 50% of saturation of dissolved oxygen. Oxygen flow was adjusted so as not to cause foaming of the mixture. The oocysts were maintained under these conditions for about 72 hours. During sporulation, dissolved oxygen and pH were constantly monitored. It was observed that beginning at approximately 12 hours into the sporulation process there was a decrease in the percent saturation of dissolved oxygen (increased oxygen consumption) followed by an increase in pH and a return of dissolved oxygen to previous levels. These changes in dissolved oxygen and pH were found to be reliable indicators of sporulation. Examination of oocysts following these changes showed a high degree of sporulation. Tangential Flow Filtration / Sterilization Following sporulation, the sporulated oocysts were concentrated by tangential flow filtration. After the system was assembled about 2 to 4 liters of cold domestic water was run through the system to check for leaks, the concentrated sporulated oocysts medium was then placed into the retentate vessel of the filter unit. Domestic cold water was added to adjust the retentate vessel volume to the desired operating level and also maintain less than a desired amount of solids. The control of the diaphragm pump was set to give the desired flow and then the pump was started. The permeate flow valve was then opened after substantially all of the bubbles were removed from the membranes and a steady flow was established across the membrane. The sporulated oocysts are to be retained in the system and not the permeate. Sporocyst isolation Methods for isolation of sporocysts are well known to those skilled in the art and will only be briefly described herein. Sporulated oocysts are diluted to a concentration of about 2 x 10.sup.6 Docket No.: HVJ-009PC / ml in PBS. One ml of the 2 x 10.sup.6 / ml suspension of sporulated oocysts is added to 1.5 ml microfuge tubes containing 125 .mu.l of 0.5 mm glass beads. The tubes containing the sporulated oocysts and the glass beads are vortexed at high speed for five minutes and then chilled on ice. Following vortexing, the supernatant is removed and saved. The glass beads are then washed with PBS and the supernatant saved and combined with the supernatant obtained after vortexing. The supernatant is then centrifuged at about 9020 x g for about two to three minutes and the supernatant discarded. The pellet is resuspended in 0.2 ml of PBS. The concentration of sporocysts per ml is then determined by using a hemacytometer or other method of determining cell number. III. Hybrid Protocol for Oocyst Identification and Quantitation The disclosure provides a hybrid protocol that allows for the identification and quantitation of viable Eimeria oocysts in a mixed sample that comprises a plurality of Eimeria species. The hybrid protocol combines means for determining the number of viable oocysts relative to non- viable oocysts with a digital PCR reaction that allows for determining the proportion of viable oocysts for each Eimeria species in the sample. Accordingly, in one aspect, the disclosure provides a method for determining the number and proportion of viable Eimeria oocysts relative to non-viable Eimeria oocysts in a sample comprising a plurality of Eimeria species, the method comprising: determining the number of viable Eimeria oocysts relative to non-viable Eimeria oocysts in the sample; extracting DNA from the viable Eimeria oocysts; subjecting the extracted DNA to digital PCR (dPCR) reactions producing amplicons specific for each Eimeria species in the sample; and determining the proportion of viable Eimeria oocysts for each Eimeria species in the sample. In another aspect, the disclosure provides a method for determining the viability of Eimeria oocyst samples relative to non-viable oocyst samples recovered at different stages of vaccine production comprising: determining the number and proportion of viable oocysts relative to non-viable oocysts in each of a plurality of oocyst samples recovered following a different stage of vaccine production; Docket No.: HVJ-009PC extracting DNA from sporocysts excysted from the viable oocysts and subjecting the extracted DNA to dPCR reactions producing amplicons specific for each of the protozoa species in the preparation; determining the copy number of PCR products corresponding to each of the one or more species; and determining the number and proportion of viable oocysts relative to non-viable oocysts for each species based on the copy number of amplicons corresponding to each species, wherein the vaccine preparation is derived from a purified and sporulated oocyst sample. An embodiment of the above method comprises the following: (a) staining oocyst samples from a vaccine preparation recovered from each of a plurality of different stages of vaccine production, wherein each oocyst sample comprises a plurality of sporocyst- forming protozoa species, and wherein each oocyst sample is stained with a vital dye; (b) determining the number and proportion of viable oocysts relative to non-viable oocysts in each oocyst sample, wherein the number and proportion of viable oocysts relative to non-viable oocysts in each oocyst sample is determined by correlating the extent of staining in each oocyst sample; (c) preparing viable oocyst samples from each oocyst sample in step (b), wherein the viable oocysts in each oocyst sample are separated and isolated away from the non-viable oocysts; (d) excysting sporocysts from each viable oocyst sample in step (c); (e) extracting DNA samples from the excysted sporocysts in step (d); (f) subjecting each DNA sample in step (e) to dPCR reactions comprising a plurality of SCAR primer pairs, wherein each SCAR primer pair is specific for a different sporocyst-forming protozoa species; (g) synthesizing SCAR amplicons produced from each dPCR reaction in step (f); (h) determining the copy number for each SCAR amplicon by comparison to standard curves generated from known amounts of polynucleotides corresponding to each SCAR amplicon; and (i) determining the proportion and number of viable oocysts corresponding to each of the protozoa species, based on the results in steps (b) and (h), wherein the vaccine preparation is derived from a fecal sample, and Docket No.: HVJ-009PC wherein the different stages of vaccine production are selected from the group consisting of an initial sample collection stage, an oocyst preparation stage, a species blending stage, a packaging stage, and a combination thereof. In an embodiment, steps (a)-(i) are repeated for a portion of the vaccine preparation tested at different dates relative an expiration date corresponding to the vaccine preparation. Various aspects of the methods of the disclosure are described in further detail below, as well as in the Examples. Staining Sporocysts are stained by treatment with a sufficient 20 quantity of at least one vital stain to produce a detectable signal. Any dye or combination of dyes capable of distinguishing between viable (live) and non-viable (dead) cells can be used. In a preferred embodiment, the dye is propidium monoazide (PMA), the use of which to stain viable vs. non-viable cells is described in detail in the Examples. In one embodiment, dyes that measure viability on the basis of membrane integrity can be used. Especially useful are nucleic acid staining dyes, such as nucleic acid intercalating dyes. One example, is heterocyclic dyes that are excluded by cells with intact membranes such as the phenanthridinium dyes propidium iodide, ethidium bromide, ethidium monoazide and ethidium bromide homodimer. Another example of a dye that can be used to assess viability based on membrane integrity is calcfluor white (CFW), the disodium salt of 4,4'-bis(4-anilino-bis-diethyl amino-s-triazin- 2-ylamino)-2,2'-stilbene disulphonic acid. Viability can also be determined using dyes that measure intracellular enzymatic activity such as carboxy fluorescein diacetate or calcien-AM. Indicators of membrane energization can likewise be used to determine viability. Examples of dyes which provide a measure of membrane energization are rhodamine 123, carbocyanine dyes and dyes of the oxonol family. Other stains suitable for determination of cell viability will be readily apparent to those skilled in the art. Optionally, a combination of stains can be used. For example, a combination of vital stains can be used so that both live and dead protozoa are stained. The live and dead protozoa are then differentiated on the basis of, for example, color or emission wavelength. An example of such a Docket No.: HVJ-009PC combination is propidium iodide and carboxy fluorescein diacetate. Another approach is to combine a vital stain with a dye which stains all protozoa regardless of viability to aid, for example, in counting the number of protozoa present. An example of such a combination would be DAPI (4'-6-diamidino- 2-phenylindole) and propidium iodide. Other possible combinations of stains will be readily apparent to those skilled in the art. In one embodiment, protozoa are stained with a fluorescent nucleic acid staining dye (propidium monoazide (PMA), PMAxx, ethidium monoazide bromide (EMA), propidium iodide (PI) or ethidium bromide). Factors affecting staining conditions include, but are not limited to, such things as the stain used, the detection method employed, the species of sporocyst stained, temperature, the staining solution, and other factors known in the art. Optimal staining conditions can readily be determined by one skilled in the art without undue experimentation. For example, the concentration of PMA can range from 5-100 .mu.M for fluorescence microscopy and flow cytometry. The concentration of ethidium bromide can range up to about 100 .mu.g / ml. When detection is by fluorescence microscopy, the concentration of ethidium bromide is preferably between about 5 .mu.g / ml and 75 .mu.g / ml, more preferably between about 10 .mu.g / ml and 50 .mu.g / ml and more preferably still about 20 .mu.g / ml. When detection is by flow cytometry, the concentration of ethidium bromide is preferably less than 25.mu.g / ml, more preferably between about 1.mu.g / ml and about 15 .mu.g / ml, even more preferably between about 2 .mu.g / ml and about 10 .mu.g / ml, more preferably still between about 3.mu.g / ml and about 7.mu.g / ml, and most preferably about 5.mu.g / ml. Likewise, staining temperature can range from about 4. degree. C. to 43. degree. C. It will be appreciated by those skilled in the art that the staining time will vary with the temperature and concentration of stain used, but that staining times can vary from approximately 1 minute to 12 hours. Staining solutions will vary with the stain used. In the present invention, the staining solution preferably contains taurodeoxycholic acid, although other bile salts could be substituted. The concentration of taurodeoxycholic acid can vary from about 0.4% to 10% and preferably is about 4%. Without being limited by theory, taurodeoxycholic acid is thought to increase the permeability of the sporocyst coat without destroying the micropyle cap. Thus, any non-toxic material which increases the permeability of the sporocyst coat without destroying the micropyle cap could be utilized. In another embodiment, sporocysts are stained for approximately 10 minutes at 41. degree. C. in a Docket No.: HVJ-009PC solution containing between about 5 and 20 .mu.g / ml ethidium bromide and about 4% taurodeoxycholic acid. Counting Once stained, it is necessary to count the number of stained and unstained sporocysts in the sample. Any method capable of detecting the presence of a dye in stained protozoa can be used, and many such methods will be apparent to those skilled in the art. For example, and without limitation, when a fluorescent dye is used, a known number of stained sporocysts can be placed in multi-well plates and the amount of fluorescence determined using a fluorescence plate scanner. The number of stained (dead) sporocysts can be determined by constructing a standard curve using known numbers of stained, dead sporocysts. In like manner, the number of stained sporocysts can be determined by fluorometry or fluorescence spectrophotometry. In one embodiment of the invention, the number of stained and unstained sporocysts is determined using a fluorescence microscope. Sporocysts, for example, can be placed in a hemacytometer slide or wells of a multi-well plate and the total number of sporocysts and the number of stained sporocysts determined. Alternatively, the images obtained through the fluorescence microscope can be captured by image processing software. The software can then be used to, for example, record the location of each sporocyst in the field and then record if that sporocyst fluoresces to calculate the percentage of dead sporocysts in the sample. In yet another alternative, the image analysis system can be combined with a computer controlled microscope stage so that the computer moves the stage to search the slide containing the sample and based on predetermined parameters locates and counts the sporocysts. The process is then repeated under conditions in which only fluorescing cells are counted. The results are then used to calculate the percentage of fluorescing and so dead sporocysts. Since the sum of the percent fluorescing (dead) and non-fluorescing (live) sporocysts is assumed to equal 100%, once the percent dead sporocysts is determined, the percent live sporocysts in the sample can be calculated by subtraction. As shown in Example 3, by back calculation, the results obtained can be used to determine the percent dead in the original sample of sporulated oocysts. In another embodiment, the number of stained and unstained sporocysts is determined by use of a flow cytometer. The exact data settings used to differentiate live and dead protozoa will vary Docket No.: HVJ-009PC with factors such as the flow cytometer used, the dye(s) used and the species of protozoa to be analyzed and can be readily determined by those skilled in the art without undue experimentation. Data parameters can be set to differentiate live and dead protozoa on the basis of multiple determinants. For example, when multiple stains are used, the cytometer can be set to acquire data on multiple fluorescence parameters as well as size and density. The data acquired can then be combined to differentiate between live and dead protozoa on the basis of different colors of fluorescence and eliminate interference due to debris or organisms that differ in size from the protozoa of interest. In a preferred embodiment, the electronic gating of the flow cytometer is set so that only intact sporocysts of the species of protozoa of interest are counted. Data parameters for fluorescence intensity are set using unstained controls such that approximately 95% of the unstained sporocysts are below the threshold to be marked as a stained sporocyst and are recorded as an unstained event, for example, marked M1. The remaining stained sporocysts whose fluorescence intensity exceeds the M1 threshold are marked as stained sporocysts (stained event), for example M2, such that the total of M1 and M2 equals the total number of sporocysts counted. By construction of a single-parameter histogram, the percentage of stained and unstained sporocysts can be determined. Alternatively, the fluorescence threshold to mark an event can be set such that approximately 95% of all stained sporocysts exceed the set threshold, while all sporocysts whose fluorescence intensities are below the threshold are marked as unstained. If the flow cytometer is so equipped, sporocysts can be sorted based on their fluorescence intensity. Digital PCR The second aspect of the hybrid protocol is the use of digital PCR (dPCR) to allow for determining the proportion of viable Eimeria oocysts for each Eimeria species in the sample. As used herein, the term “digital PCR” refers to a polymerase chain reaction (PCR) method that is capable of quantifying the resultant PCR products. PCR primers are used that produce amplicons that are specific for each of the protozoa species in the preparation. As used herein, the term “amplicon” refers to the PCR product that is produced using the PCR primers. Quantification of the amplicons produced by the dPCR reaction allows for determining the proportion of viable Eimeria oocysts for each Eimeria species in the sample. Docket No.: HVJ-009PC Detailed protocols for use of dPCR with a mixed Eimeria sample are described in the Examples. In embodiments, the dPCR step uses Sequenced Characterized Amplified Region (SCAR) primers specific for each of the Eimeria species in the sample. Non-limiting examples of suitable SCAR primers are shown in Table 1 (SEQ ID NOs: 1-9) and Table 8 (SEQ ID NOs: 10-30). Following DNA extraction and dPCR (e.g., with SCAR primers), amplicons specific for each Eimeria species in the mixed sample are generated and can then be quantitated. Accordingly, in aspects, the method comprises steps including: subjecting the DNA extracted from the sample to species-specific digital PCR (dPCR) reactions comprising sequenced characterized amplified region (SCAR) primers specific for each of the one or more sporocyst-forming protozoa species; synthesizing species-specific SCAR amplicons from the dPCR reactions; determining the copy number of each species-specific SCAR amplicon produced in the dPCR reactions; and determining the number and proportion of sporulated oocysts relative to unsporulated oocysts for each of the one or more sporocyst-forming species in the mixed population, based on the results in the steps above. In embodiments, the SCAR primers comprise two or more nucleotide sequences selected from the group consisting of SEQ ID NOs: 1-9. In other embodiments, the SCAR primers comprise two or more nucleotide sequences selected from the group consisting of SEQ ID NOs: 10-30. In embodiments, the copy number of each species-specific SCAR amplicon is determined by comparison to a standard curve generated from known amounts of polynucleotides corresponding to each of the species-specific amplicons. Methods for generating such standard curves from known amounts are well-established in the art. In embodiments, the dPCR assays are corrected for DNA extraction efficiency, PCR efficiency and / or SCAR amplicon copy number, wherein the mixed sample and the SCAR pDNA for each species are subjected to a SCAR dPCR assay, and wherein correction factors for each assay are determined to calculate the original concentrations of live sporulated oocysts per species. Approaches for correcting dPCR results for DNA extraction efficiency, PCR efficiency and SCAR amplicon copy number are well-established in the art. Docket No.: HVJ-009PC IV. Definitions As used herein, the term "oocyst" refers the dormant life-cycle stage of a coccidial protozoan having a tough outer coat which protects the oocyst from environmental insult, but also makes it difficult to manipulate the oocyst in staining procedures. As formed, the oocyst is not capable of infection and may also be referred to as an "unsporulated oocyst." Oocysts are found in the intestine of animals following release from infected cells and are eliminated in the excreta. The term "sporulated oocyst" refers to an oocyst which has undergone maturation naturally or through artificial manipulation such that the sporulated oocyst is capable of infecting a susceptible host. During maturation, a multiplicity of sporocysts, each with its own outer shell or case, develops within the oocyst creating a "cyst within a cyst." For example, in E. tenella, a sporulated oocyst contains four sporocysts, each with their own outer shell or case. In the case of the sporocyst the outer shell / case is semi-permeable with properties similar to a eukaryote cell membrane, which makes them easier to manipulate for staining purposes. The term "sporocyst" means a life-cycle stage of a coccidial protozoan having an outer coat or case containing a multiplicity of sporozoites which are the ultimate infective agent of the protozoan. In the instance of E. tenella, each sporocyst contains two sporozoites. The term "sporocyst-forming protozoa" refers to protozoa which have as part of their life cycle, at minimum, a non-infective oocyst stage that is shed by infected animals, a sporulated oocyst stage, and a sporocyst stage. The terms "encysted protozoa", "encysted oocyst" and "encysted sporocyst" are used with reference to organisms which are within a cyst or have their own outer coat or shell. The terms "excysted protozoa" and "excysted sporozoite" both refer to an organism in which the outer shell or coat has been removed either naturally or through artificial means. As used herein, the terms “digital PCR” and dPCR are used with reference to a nucleic acid amplification method for the quantitation and analysis of target nucleic acid sequences by partitioning the reaction into a sufficient number of reaction subvolumes so that the target is in limiting dilution, i.e., producing a sufficient number of reaction subvolumes with zero target copies so as to allow the application of Poisson statistics. Docket No.: HVJ-009PC V. Equivalents and Scope Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments in accordance with the disclosure described herein. The scope of the present disclosure is not intended to be limited to the foregoing Description, but rather is as set forth in the appended claims. In the claims, articles such as “a,” “an,” and “the” mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process, unless indicated to the contrary or otherwise evident from the context. Throughout the Description and Claims, embodiments are provided in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. Throughout the Description and Claims, embodiments are provided in which more than one or all of the group members are present in, employed in, or otherwise relevant to a given product or process. Throughout the Description and Claims, use of the term “comprising” is intended to be open and contemplates or permits the inclusion of additional elements or steps. Where ranges are given, endpoints are included. Furthermore, it is to be understood that, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of the disclosure, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. In addition, it is to be understood that any particular embodiment of the present disclosure that falls within the prior art may be explicitly excluded from any one or more of the claims. Since such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the compositions of the disclosure (e.g., any small molecule; any method of production; any method of use; etc.) can be excluded from any one or more claims, for any reason, whether or not related to the existence of prior art. Docket No.: HVJ-009PC All cited sources, for example, references, publications, databases, database entries, and art cited herein, are incorporated into this application by reference, even if not expressly stated in the citation. In case of conflicting statements of a cited source and the instant application, the statement in the instant application shall control. The disclosure is further illustrated by the following non-limiting examples. It is to be understood that the foregoing description and following examples are intended to illustrate and not limit the scope of the present disclosure, which is defined by the scope of the appended claims. EXAMPLES Example 1: Development of Hybrid Protocol for Quantitation of Eimeria Oocysts A hybrid method based on a detergent-enhanced staining method, a volumetric viability assessment by flow cytometry and a molecular determination of the species composition of sorted viable oocysts was developed to determine the number of viable oocysts per species in a mixed sample containing oocysts from the turkey coccidia species, E. gallopavonis, E. adenoides and E. meleagrimitis. SCAR primers specific for these turkey coccidia species (Vrba et al., Int. J. Parasitol., 2014) were tested and validated for their applicability in qPCR and dPCR. Specific and sensitive SCAR dPCR quantification was performed with SCAR pDNA standards to correctly quantify the coccidia species in the mixed sample. In addition, PMA live-dead staining was carried out in combination with a detergent-enhanced staining method to determine the number of viable oocysts per species in the mixed oocyst sample. I. Materials and methods Monoculture parasite samples E.ga-230119(MS) and EGA-USUS-28May19 Eade-29May19(MS) and EAD-USUS-04Aug20 Emt-290119(MS) and EMT-USUS-21Jun20 DNA extracts Ega-020217(MS) Ead-1603147(MS) Docket No.: HVJ-009PC Emt-131216(MS) DNA extraction DNA extractions were performed following an optimized Eimeria DNA extraction protocol based on standard procedures in the art. Development of SCAR plasmid DNA - SCAR amplification Fifty nanogram (ng) of the Eimeria spp. DNA extracts were subjected to a PCR reaction containing 12.5 µL 2× Q5 high fidelity MM (NEB), 1.25 µL forward- and 1.25 µL reverse primer (final concentration of 500 ƞM) in a total reaction mixture of 25 µL. PCR was performed on a Bio- rad T100 thermal cycler with a 30-second activation step at 98°C, 32 amplification cycles (5 min. at 98°C, 15” at 63°C and 10 min. at 72°C) and a 2-minute final elongation at 72°C. PCR products were checked by 1% agarose gel electrophoresis and purified using a QIAquick PCR Purification Kit. Development of SCAR plasmid DNA - A-tailing Since Phusion DNA polymerase has 3´→ 5´ exonuclease activity and generates blunt- ended products, an extra incubation step with Taq-polymerase was performed to create 3´ A- overhangs which are essential for TOPO TA cloning. For this, 10 µL of purified PCR product was incubated with 20 µL GoTaq G2 mastermix (Promega) and 10 µL water for 20 minutes at 72°C. The A-tailed product was purified using a QIAquick PCR Purification Kit (Qiagen). Development of SCAR plasmid DNA - Eimeria spp. SCAR amplicon in pCR2.1 vector Ligation of the A-tailed SCAR amplification products of the different Eimeria spp. into the pCR2.1 vector (Invitrogen) was performed using the TA Cloning®Kit according to the manufacturer’s instructions. The appropriate amount of insert was mixed with T4 ligase buffer, 50 ng pCR2.1 vector and T4 ligase and incubated for 2 hours at 20°C in a Bio-rad T100 thermal cycler. Development of SCAR plasmid DNA - Transformation in NEB10-beta competent bacteria Two µL of the ligation product was added to 50 µL of NEB10-beta competent bacteria and transferred to an ice-cold 0.2 cm Gene Pulser cuvette (Bio-rad) for electroporation (25 µF, 200 Ω and 2.5 kV) in a Bio-rad Gene Pulser. Transformed bacteria were transferred to a 15 mL Falcon tube together with 950 µl SOC outgrowth medium and incubated for 1 h at 37°C and 250 rpm shaking, after which 100 µL and 200 µL was plated on LB-agar containing 100 µg / mL ampicillin and incubated overnight at 37°C. Docket No.: HVJ-009PC Development of SCAR plasmid DNA - Colony PCR using the SCAR qPCR assay Eight colonies per species were subjected to the SCAR qPCR. A 20 µL TaqMan reaction mixture contained 10 µL of 2× SensiFAST Probe No-ROX Mix (Bioline), 1 µL diluted colony and optimized primer and probe concentrations as shown in Table 1. Table 1. Primers for SCAR qPCR. ugh MGM_SCAR_P, are also shown in SEQ ID NOs: 1-9, respectively. All samples were tested on a Roche Lightcycler 96 real-time PCR system using a 5-minute activation step at 95°C and 45 amplification cycles under optimized conditions (10” at 95°C and 30” at 60°C). Three positive colonies per species were sent for Sanger Sequencing (same primers that were used for amplification) on an Applied Biosystems 3730XL DNA. Analyzer at the Neuromics Support Facility, University of Antwerp and analyzed using SnapGeneTMand NCBI BLAST. SCAR qPCR assays The TaqMan reaction mixture contained 10 µL of 2x SensiFAST Probe No-ROX Mix (Bioline), 2 µL diluted sample and optimized primer and probe concentrations. All samples were tested on a Roche Lightcycler 96 real-time PCR system with a 5 minute activation step at 95°C and 45 amplification cycles (10” at 95°C and 10” at 60°C). SCAR dPCR assays The dPCR reaction mixture contained 3.75 µL 4x Probe PCR Master Mix, 1.2 µL SCAR forward primer, 1.2 µL SCAR reverse primer, 0.6 µL probe, 0.04 µL NotI_HF and 3.21 µL water. 10 µL of MM was added to a 96 well plate after which 5 µL of sample was added. 12.5 µL of the Docket No.: HVJ-009PC MM-sample mixture was transferred to a QIAcuity Nanoplate 8.5k 96-well and tested on the QIAcuity dPCR system with a 2-minute activation step at 95°C and 40 amplification cycles (15” at 95°C and 30” at 60°C). Imaging conditions were set at Exposure 700 ms and Gain 8. PMA viability staining PMA dye (propidium monoazide, Biotium) is a viability stain that cannot enter intact cells. Upon penetration of membranes of dead cells, the dye forms covalent bonds with dsDNA upon photoactivation. Hence, PMA-staining will differentiate between live and death cells during a PCR reaction by inhibiting DNA-amplification from death cells. Samples were incubated together with 0.5% Triton-X100 and 100 μM PMA for 4 min at 61°C in a thermomixer at 300 rpm completed with 6 min at room temperature (RT). Next, the samples were photoactivated by exposure to light for 20 min (λ: 465-475 nm, PMA-LiteTMLED photolysis device, VWR). Flow cytometry PMA-stained oocysts were washed after photoactivation, and pellets were resuspended in PBS. Samples were analysed using a 2.0 neutral density filter and an agitation of 100 rpm. Selection of the oocyst population was based on SSC-A and FSC-A and followed by gating based on autofluorescence (λex: 405 nm – λem: 528 / 45 nm). PMA-positive and -negative populations were detected using the yellow-green laser and an associated filter set (λex: 561 nm - λem: 613 / 18). To avoid underestimation of viability, the background signal of unstained samples was determined. Data were analysed using FlowLogicTMsoftware. Fluorescence microscopy Oocysts were washed after photoactivation and observed by fluorescence microscopy (λex:546 / 12 nm - λem:575-640 nm; Zeiss Axio Observer.Z1 Inverted Phase Contrast Fluorescence Microscope). II. Specificity of SCAR qPCR assays. A 5-fold serial dilution was made of the DNA extractions from E. gallopavonis (Ega- 020217(MS)), E. adenoides (Ead-1603147(MS)) and E. meleagrimitis (Emt-131216(MS)). In addition, a 1 / 5 diluted sample with a mixture of all three species was made by combining 1 amount of every species DNA extract with 2 amounts of water (1-1-1-2). The monoculture serial dilutions and the mixture sample were subjected to the SCAR qPCR assays specifically targeting the Docket No.: HVJ-009PC individual species. As shown in FIG. 1, SCAR qPCR proved to be specific for all species, although some cross-species amplification products were seen for E. gallopavonis and E. adenoides but in such low amounts so as to not interfere with specific quantification. Comparison of the 1 / 5 diluted monoculture and 1 / 5 diluted mixture confirmed this observation with highly comparable Cq values. Good PCR efficiencies calculated on monoculture serial dilutions ensure accurate quantification. III. Eimeria spp SCAR amplicon cloning in the pCR2.1 vector Fifty nanogram (ng) of the Eimeria spp. DNA extracts were amplified using SCAR primers and cloned into a pCR2.1 vector (FIG.2, Table 2). Three positive control plasmids harboring the SCAR target sequence of each Eimeria spp. were identified by Sanger sequencing. Sequences where identical for all three clones suggesting a well conserved molecular target. Sequences did not align Eimeria spp. using NCBI BLAST, meaning that SCAR sequences have not yet been uploaded into the Genbank database. pDNA standards were measured very accurately with a Quantus fluorometer after which they were diluted and aliquoted into fixed amounts of copies / µL based on molecular weight and Avogrado’s number, and stored at -80°C. Table 2. SCAR sequences cloned into the control plasmids. IV. Sensitivity of SCAR qPCR and dPCR assays Oocysts (n= 10,000) of each species were sorted with a FACSMelody in technical duplicate after which DNA was extracted using our optimized protocol. gDNA and pDNA standards were subjected to SCAR PCR primer / probe sets on real time-PCR (qPCR) and digital PCR (dPCR) for determination of sensitivity. Comparable results were obtained for technical duplicates, ensuring a Docket No.: HVJ-009PC robust DNA extraction technique. For gDNA standards, both qPCR and dPCR showed a limit of detection (LoD) of about 3-55 parasites per reaction, corresponding to 1 – 7 sporulated oocysts. dPCR on E. gallopavonis gDNA resulted in a 3× higher copies / µL than for the other species, possibly due to a SCAR marker that is present 3× more in the genome (Table 3). Table 3. Individual qPCR and dPCR data for gDNA standards. t 1 sporulated oocyst. PCR efficiencies for pDNA standards were very good both in qPCR (Table 4) and dPCR (Table 5), enabling the use of correction factors required for accurate and sensitive quantification in mixed samples. For dPCR, pDNA standards resulted in the expected copies / µL, validating the novel dPCR technique as reliable for quantification (Table 5, FIG.3). Table 4. Individual qPCR data for pDNA standards.

[0002] Docket No.: HVJ-009PC

[0003] Docket No.: HVJ-009PC Table 5. Individual dPCR data for pDNA standards. . Monoculture oocyst samples of all three species were PMA-stained in combination with the optimized permeabilization technique developed in the chicken Eimeria project. PMA was able to penetrate and bind DNA of all species as seen in ‘untreated’ and ‘heat-killed’ epifluorescence images (FIG. 4). No background signal was observed in unstained controls. These observations were confirmed by FACSMelody where a clear shift in median fluorescence intensity (MFI) was observed in heat-killed samples compared to unstained and untreated samples (FIG. 5), indicating that PMA is suitable for viability staining of oocysts from the three turkey coccidia species. As previously observed, occasional wall staining was observed both in fluorescence microscopy and FACSMelody. These wall- stained oocysts are scored viable since PMA is not able to cross the rigid oocyst wall. PMA staining resulted in a volumetric determination of ± 50% viable oocysts in comparison with the original amount Docket No.: HVJ-009PC for E. adenoides (EAD-USUS-04Aug20) and E. meleagrimitis (EMT-USUS-21Jun20), whereas only 32 % of original oocysts remained viable for E. gallopavonis (EGA-USUS-28May19) (Table 6). Table 6. Volumetric determination on PMA-stained untreated samples, EAD-USUS- 04Aug20, EGA-USUS-28May19, and EMT-USUS-21Jun20. Example 2. Use of Hybrid Protocol to Quantitate Viability of Mixed Species Eimeria Vaccines The methodology in Example 1 was applied to obtaining a volumetric species-specific viability determination of oocysts in a multivalent vaccine. To further optimize the hybrid protocol, an absolute quantitative molecular assay was introduced, i.e. digital PCR (dPCR). In this study, plasmid DNA containing the SCAR DNA sequences of the 6 Eimeria spp. were developed as molecular standards. In order to account for DNA extraction efficiency, PCR efficiency and gene copy number, the molecular assays were performed on mixed oocyst samples that were composed by sorting of monocultures. To validate the complete hybrid method, the protocol was applied to various MMAT 1000 and 5000 vaccine batches with different expiry dates. Additionally, an initial analysis of the viability of attenuated and parent strains was performed by flow cytometry.

[0004] Docket No.: HVJ-009PC I. Materials and methods. Table 7. Samples. Parent and Monocultures MMAT vaccines attenuated 21 1 1 1 1 1 SCAR plasmid DNA (pDNA) was obtained as previously described in Example 1, using SCAR primers adopted from Vrba et al. (2010) (Table 8). The concentration of the purified circular pDNA was measured with a Quantus Fluorometer (Promega) using the QuantiFluor dsDNA sample kit (Promega). SCAR pDNA was diluted in TE buffer to 0.105 ng / µL, which corresponds to 0.5 × 108target copies using the in silico determined molecular weight, plasmid length and Avogrado’s number. pDNA samples were kept at -80°C. A 10-fold serial dilution was made for each Eimeria spp. and 5 µL was subjected to the corresponding SCAR dPCR assays at least in duplicate.

[0005] Docket No.: HVJ-009PC Table 8. Overview of SCAR primers for chicken Eimeria spp. PCR reaction primer and probe Eimeria target Sequence of primers and probesi n e also shown in SEQ ID NOs: 10-30, respectively. dPCR assay correction factors In order to be able to account for the DNA extraction efficiency, PCR efficiency and gene copy number in mixed MMAT samples, live E. mitis, E. acervulina and E. tenella oocysts from monocultures were combined in various proportions by sorting using the FACS Melody apparatus. Mixed samples were composed in triplicate and consisted of 35,000 viable oocysts with different proportions of the 3 species (Table 9). DNA of the mixed oocyst samples was extracted as described in the detailed description in paragraph 0038-0039. A dPCR assay was performed with the mixed oocyst samples and their respective pDNA standards. All calculations were performed on log10- transformed data. The theoretically expected and experimentally determined numbers of pDNA copies Docket No.: HVJ-009PC (dPCR assay) were plotted and a linear regression was performed in Graphpad Prism 9. For the mixed samples with different species proportions the same analysis was performed for each Eimeria spp., using the fixed slopes of the pDNA standards. The difference in intercept (∆Yintercept) between the mixed samples and pDNA standard curve was used as dPCR assay correction factor to account for DNA extraction efficiency, PCR efficiency and gene copy number. Table 9 Mixed samples with various proportions of viable E. mitis, E. acervulina and E. tenella prepared by sorting. Samples were prepared in triplicate. MIAT mix Viable oocysts / spp. sorted o 11,600 E. mitis Viability of MMAT 1000 and MMAT 5000 vaccines of different shelf lives (a) Volumetric viability analysis and sorting of viable oocysts by FACS Melody. The viability of this vaccine was analyzed in triplicate as described herein. The viable gate was analyzed microscopically for purity and sporulation and this was taken into account in the calculation of the total volumetric viability. The volumetric number of oocysts of both viable E. maxima and the combination of the other 3 Eimeria. spp. was determined by FACSMelody. (b) Digital PCR Samples consisted of 35,000 viable oocysts, containing E. mitis, E. acervulina and E. tenella. DNA extraction of viable sorted oocysts and SCAR dPCR assays were performed as described herein. (c) Species-specific volumetric viability calculations Docket No.: HVJ-009PC In order to quantify the species-specific volumetric viability for E. mitis, E. acervulina and E. tenella, the proportion of each species within the viable population gate should be determined. After a dPCR assay, thresholds were determined (1D scienceplot) based on the non-template controls for each species. Since samples were analyzed in duplicate, the averages of both outcomes per sample were used in further calculations. The outcomes of the dPCR are presented as the number of copies of the targeted gene per µL. Because the reaction mixture consists of 5 µL DNA and 10 µL master mix, the number of SCAR copies / µL detected by dPCR were multiplied by 3 to obtain the number of copies / µL in the original DNA sample. The number of copies / µL were log10-transformed for all samples. The correction factors for each Eimeria spp. (described in dPCR assay correction factors as ∆Yintercept) were added to the log10-transformed number of copies / µL. Next the values were inverse- log10-transformed and divided by 8 to account for the 8 sporozoites in an oocyst. The proportion of E. mitis, E. acervulina and E. tenella oocysts in each sample was calculated. These proportions were then applied to the volumetric viability of the mixed gate to obtain species-specific volumetric viabilities. Viability of attenuated and parent strains In order to determine the initial viability of attenuated and parent strains, samples were analyzed in triplicate as described herein. Briefly, samples were incubated at least overnight in 0.05% Tween 80. To stain oocysts, samples were incubated for 4 minutes at 61°C completed with 6 minutes at RT in the presence of 100 µM PMA, 0.5% Triton X-100 and 0.05% Tween 80 before photoactivation. Samples were analysed by FACS Melody. Selection of the oocyst population was based on SSC-A and FSC-A and followed by gating based on autofluorescence (λex: 405 nm - λem: 528 / 45 nm). PMA-positive and -negative populations were detected using the yellow-green laser and an associated filter set (λex:561 nm - λem:613 / 18). Viabilty was determined in triplicate. The viable gate was analyzed microscopically for purity and sporulation and this was taken into account for calculation of the volumetric viability. pDNA standards In order to validate the dPCR technique and to be able to include positive controls for all Eimeria spp. in every dPCR assay, SCAR pDNA standards were developed for all Eimeria spp.. As shown in FIG.6, linear regressions of the number of SCAR copies per µL detected by the dPCR assay compared to the theoretically expected number of SCAR copies per µL show good PCR efficiencies. Docket No.: HVJ-009PC This enables an accurate species-specific quantification by dPCR and including pDNA standards. Accordingly, these pDNA standards validated the dPCR method. dPCR assay correction factors In order to be able to correct dPCR assays for DNA extraction efficiency, PCR efficiency and SCAR copy number, mixed oocyst samples (Table 8) and a 10-fold dilution of SCAR pDNA of the relevant species were subjected to a SCAR dPCR assay (FIG. 7). Slopes are very similar for the 3 Eimeria spp. but there seems to be a difference between the ∆Yintercept of E. mitis and E. acervulina and the ∆Yintercept of E. tenella. This could be explained by a difference in SCAR copy number in E. tenella, which is also observed by NCBI, where a nucleotide BLAST of the SCAR E. tenella amplicon results in three 100 % hits in chromosome 7 (sequence ID: HG994967.1, positions: 42788 to 42887 / 47596 to 47695 / 52404 to 52503), whereas for E. mitis- and E. acervulina only one SCAR copy is found. Viability of MMAT 1000 and MMAT 5000 vaccines with different expiry dates In order to validate the hybrid method to determine the volumetric species-specific viability in an MMAT vaccine, the hybrid method was applied to 12 MMAT 1000 and 11 MMAT 5000 vaccine batches with different expiry dates. E. maxima oocysts showed a large reduction in viability for every evaluated MMAT 1000 and 5000 vaccine batch. For other species the reduction in viability was more variable. In general, there were more remaining viable oocysts in MMAT 5000 than in MMAT 1000 vaccines with corresponding expiry dates (FIG.8 and FIGS.9A, 9B). Despite debris in the old vaccine batches that may affect the flow cytometric analysis, viable oocyst sorting and microscopic evaluation confirmed the presence of live and sporulated oocysts in all tested batches (FIG. 10). In addition, absolute SCAR copy numbers in sorted viable oocysts are very consistent across tested batches (FIG. 11). Viability of attenuated and parent strains In order to determine the viability of attenuated and parent strains of oocysts of different species, they were stained using the detergent-enhanced method and analyzed in triplicate by FACSMelody. Attenuated lines tend to show a slightly lower volumetric viability than their parent strain counterparts (FIG. 12). Small differences between attenuated and parent strains of the same Docket No.: HVJ-009PC Eimeria spp. could be observed in the forward and side scatter (Figure S2). Attenuated lines tend to give rise to a larger percentage of wall stained oocysts upon PMA staining compared to parent strains (FIG. 13). This could indicate that there is a difference in the oocyst wall composition of attenuated versus parent strain oocysts.

[0006] Docket No.: HVJ-009PC SUMMARY OF SEQUENCE LISTING SEQ ID NO: SEQUENCE

Claims

Docket No.: HVJ-009PC CLAIMS 1. A method for determining the number and proportion of viable Eimeria oocysts relative to non-viable Eimeria oocysts in a sample comprising a plurality of Eimeria species, the method comprising: determining the number of viable Eimeria oocysts relative to non-viable Eimeria oocysts in the sample; extracting DNA from the viable Eimeria oocysts; subjecting the extracted DNA to digital PCR (dPCR) reactions producing amplicons specific for each Eimeria species in the sample; and determining the proportion of viable Eimeria oocysts for each Eimeria species in the sample.

2. The method of claim 1, wherein the number of viable Eimeria oocysts relative to non-viable Eimeria oocysts is determined by staining the viable and non-viable oocysts with a vital dye.

3. The method of claim 2, wherein the vital dye is a fluorescent dye, a nucleic acid staining dye, or a propidium derivative.

4. The method of claim 3, wherein the vital dye is propidium monoazide.

5. The method of any one of claims 2-4, wherein the staining is carried out in the presence of one or more non-ionic surfactants.

6. The method of claim 5, wherein the one or more non-ionic surfactants comprise Triton X-100, Tween 80, or both.

7. The method of any one of claims 2-6, wherein the number and / or proportion of viable oocysts is based on the extent of staining by the non-viable oocysts.

8. The method of claim 7, wherein the extent of staining is determined by fluorescence microscopy.

9. The method of claim 7, wherein the extent of staining is determined by flow cytometry.Docket No.: HVJ-009PC 10. The method of any one of claims 1-9, wherein the step of extracting the DNA is carried out following isolation of the viable oocysts by fluorescence-activated cell sorting (FACS).

11. The method of any one of claims 1-10, comprising: (a) staining oocysts from a vaccine preparation comprising one or more mixed population of sporocyst-forming Eimeria species with a vital dye; (b) determining the number and proportion of viable oocysts for each of species based on the extent of staining in step (a); (c) isolating the viable oocysts from the non-viable oocysts; (d) excysting sporocysts from the viable oocysts and extracting DNA from the sporocysts; (e) subjecting the DNA extracted in step (d) to species-specific digital PCR (dPCR) reactions comprising sequenced characterized amplified region (SCAR) primers specific for each of the one or more sporocyst-forming protozoa species; (f) synthesizing species-specific SCAR amplicons from the dPCR reactions; (g) determining the copy number of each species-specific SCAR amplicon produced in step (f); and (h) determining the number and proportion of sporulated oocysts relative to unsporulated oocysts for each of the one or more sporocyst-forming species in the mixed population, based on the results in steps (b) and (g).

12. The method of any one of claims 1-11, wherein the sample comprises a plurality of different sporocyst-forming protozoa species.

13. The method of claim 11 or 12, wherein the SCAR primers comprise two or more nucleotide sequences selected from the group consisting of SEQ ID NOs: 1-9.

14. The method of claim 11 or 12, wherein the SCAR primers comprise two or more nucleotide sequences selected from the group consisting of SEQ ID NOs: 10-30.

15. The method of any one of claims 11-14, wherein the copy number of each species-specific SCAR amplicon is determined by comparison to a standard curve generated from known amounts of polynucleotides corresponding to each of the species-specific amplicons.Docket No.: HVJ-009PC 16. The method of any one of claims 11-15, wherein the dPCR assays are corrected for DNA extraction efficiency, PCR efficiency and SCAR amplicon copy number, wherein the mixed sample and the SCAR pDNA for each species are subjected to a SCAR dPCR assay, and wherein correction factors for each assay are determined to calculate the original concentrations of live sporulated oocysts per species.

17. The method of any one of claims 11-16, wherein the vital dye is a fluorescent dye, a nucleic acid staining dye, or a propidium derivative.

18. The method of claim 17, wherein the vital dye is propidium monoazide.

19. The method of any one of claims 11-18, wherein the staining in step (a) is carried out in the presence of one or more non-ionic surfactants.

20. The method of claim 19, wherein the one or more non-ionic surfactants comprise Triton X- 100, Tween 80, or both.

21. The method of any one of claims 11-20, wherein the staining in step (a) is carried out in the presence of a brief heat treatment.

22. The method of any one of claims 11-20, the extent of staining is determined by the number of non-viable oocysts.

23. The method of any one of claims 11-20, wherein the extent of staining is determined by fluorescence microscopy.

24. The method of any one of claims 11-20, wherein the extent of staining is determined by flow cytometry. 25 The method of any one of claims 1-23, wherein the lower limit of detection (LoD) for viable oocysts is 1-5 oocysts per species.Docket No.: HVJ-009PC 26. The method of claim 1, wherein the oocyst sample comprises oocysts from at least two Eimeria species selected from the group consisting of E. mitis, E. acervulina, E. tenella, E. maxima, E. necatrix, E. praecox, and E. brunetti.

27. The method of claim 1, wherein the oocyst sample comprises oocysts from E. mitis, E. acervulina, E. tenella, and E. maxima.

28. The method of claim 1, wherein the oocyst sample comprises oocysts from at least two Eimeria species selected from the group consisting of E. 29innocua, and E. subrotunda.

29. The method of claim 1, wherein the oocyst sample comprises oocysts from E. adenoeides and E. meleagrimitis.

30. The method of any one of claims 1-29, wherein the sample is a fecal sample.

31. A method for determining the viability of Eimeria oocyst samples relative to non-viable oocyst samples recovered at different stages of vaccine production comprising: determining the number and proportion of viable oocysts relative to non-viable oocysts in each of a plurality of oocyst samples recovered following a different stage of vaccine production; extracting DNA from sporocysts excysted from the viable oocysts and subjecting the extracted DNA to dPCR reactions producing amplicons specific for each of the protozoa species in the preparation; determining the copy number of PCR products corresponding to each of the one or more species; and determining the number and proportion of viable oocysts relative to non-viable oocysts for each species based on the copy number of amplicons corresponding to each species, wherein the vaccine preparation is derived from a purified and sporulated oocyst sample.

32. The method of claim 31, wherein the different stages of vaccine production are selected from the group consisting of an initial sample collection stage, an oocyst preparation stage, a species blending stage, a packaging stage, and a combination thereof.Docket No.: HVJ-009PC 33. The method of claim 31 or 32, wherein the amplicons in step (b) are sequence-specific SCAR amplicons produced by using SCAR primers specific for each of the one or more sporocyst-forming protozoa species; 34. The method of any one of claims 31-33, wherein the SCAR primers comprise two or more nucleotide sequences selected from the group consisting of SEQ ID NOs: 1-9.

35. The method of any one of claims 31-33, wherein the SCAR primers comprise two or more nucleotide sequences selected from the group consisting of SEQ ID NOs: 10-30.

36. The method of any one of claims 31-35, wherein the copy number of each species-specific SCAR amplicon is determined by comparison to a standard curve generated from known amounts of polynucleotides corresponding to each of the species-specific amplicons.

37. The method of any one of claims 31-36, wherein the dPCR assays are corrected for DNA extraction efficiency, PCR efficiency and SCAR amplicon copy number, wherein the mixed sample and the SCAR pDNA for each species are subjected to a SCAR dPCR assay, and wherein correction factors for each assay are determined to calculate the original concentrations of live sporulated oocysts per species and the original concentrations of SCAR copies per species in the mixed sample.

38. The method of any one of claims 31-37, wherein the vaccine preparation comprises oocysts from at least two species selected from E. mitis, E. acervulina, E. tenella, E. maxima, E. necatrix, E. praecox, and E. brunetti.

39. The method of claim 38, wherein the vaccine preparation comprises oocysts from E. mitis, E. acervulina, E. tenella, and E. maxima.

40. The method of any one of claims 31-37, wherein the vaccine preparation comprises oocysts from at least two species selected from E. meleagridis, E. meleagrimitis, E. gallopavonis, E. adenoeides, E. dispersa, E. innocua, and E. subrotunda.

41. The method of claim 40, wherein the vaccine preparation comprises oocysts from E. adenoeides and E. meleagrimitis.Docket No.: HVJ-009PC 42. The method of claim 31, comprising: (a) staining oocyst samples from a vaccine preparation recovered from each of a plurality of different stages of vaccine production, wherein each oocyst sample comprises a plurality of sporocyst-forming protozoa species, and wherein each oocyst sample is stained with a vital dye; (b) determining the number and proportion of viable oocysts relative to non-viable oocysts in each oocyst sample, wherein the number and proportion of viable oocysts relative to non-viable oocysts in each oocyst sample is determined by correlating the extent of staining in each oocyst sample; (c) preparing viable oocyst samples from each oocyst sample in step (b), wherein the viable oocysts in each oocyst sample are separated and isolated away from the non-viable oocysts; (d) excysting sporocysts from each viable oocyst sample in step (c); (e) extracting DNA samples from the excysted sporocysts in step (d); (f) subjecting each DNA sample in step (e) to dPCR reactions comprising a plurality of SCAR primer pairs, wherein each SCAR primer pair is specific for a different sporocyst-forming protozoa species; (g) synthesizing SCAR amplicons produced from each dPCR reaction in step (f); (h) determining the copy number for each SCAR amplicon by comparison to standard curves generated from known amounts of polynucleotides corresponding to each SCAR amplicon; and (i) determining the proportion and number of viable oocysts corresponding to each of the protozoa species, based on the results in steps (b) and (h), wherein the vaccine preparation is derived from a fecal sample, and wherein the different stages of vaccine production are selected from the group consisting of an initial sample collection stage, an oocyst preparation stage, a species blending stage, a packaging stage, and a combination thereof.

43. The method of claim 42, wherein steps (a)-(i) are repeated for a portion of the vaccine preparation tested at different dates relative an expiration date corresponding to the vaccine preparation.

44. The method of claim 42 or 43, wherein the dPCR assays are corrected for DNA extraction efficiency, PCR efficiency and SCAR amplicon copy number, wherein the mixed sample and the SCAR pDNA for each species are subjected to a SCAR dPCR assay, and wherein correction factors for each assayDocket No.: HVJ-009PC are determined to calculate the original concentrations of live sporulated oocysts per species and the original concentrations of SCAR copies per species in the mixed sample.

45. The method of any one of claims 42-44, wherein the vital dye is a fluorescent dye, a nucleic acid staining dye, or a propidium derivative.

46. The method of claim 44, wherein the vital dye is propidium monoazide.

47. The method of any one of claims 42-46, wherein the staining is carried out in the presence of one or more non-ionic surfactants.

48. The method of claim 41, wherein the one or more non-ionic surfactants comprise Triton X- 100, Tween 80, or both.

49. The method of any one of claim 42, wherein the staining is carried out in the presence of a brief heat treatment.

50. The method of any one of claims 42-49, the extent of staining is determined by the number of non-viable oocysts.

51. The method of any one of claims 42-50, wherein the number and proportion of viable oocysts is determined by fluorescence microscopy.

52. The method of any one of claims 42-50, wherein the number and proportion of viable oocysts in step is determined by flow cytometry.

53. The method of any one of claims 42-52, wherein the step of extracting the DNA is carried out following isolation of the viable oocysts by flow cytometry.

54. The method of any one of claims 42-53, wherein each oocyst sample comprises species belonging to the Apicomplexa phylum.Docket No.: HVJ-009PC 55. The method of claim 54, wherein each oocyst sample comprises species belonging to a genus selected from the group consisting of Eimeria, Isospora, Cryptosporidium, Toxoplasma and combinations thereof.

56. The method of claim 55, wherein each oocyst sample comprises Eimeria species.

57. The method of claim 56, wherein each oocyst sample comprises Eimeria species selected from the group consisting of E. mitis, E. acervulina, E. tenella, E. maxima, E. necatrix, E. praecox, and E. brunetti.

58. The method of claim 57, wherein each oocyst sample comprises a mixed population of E. mitis, E. acervulina, E. tenella, and E. maxima.

59. The method of claim 58, wherein each oocyst sample comprises Eimeria species selected from the group consisting of E. meleagridis, E. meleagrimitis, E. gallopavonis, E. adenoeides, E. dispersa, E. innocua, and E. subrotunda.

60. The method of claim 59, wherein each oocyst sample comprises a mixed population of E. adenoeides and E. meleagrimitis.

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