Evaluation of autofluorescent Eimeria maxima oocysts as a potential indicator of non-viability when enumerating oocysts

Evaluation of autofluorescent Eimeria maxima oocysts as a potential indicator of non-viability... Abstract Aging, poor oxygenation, or improper storage temperature can lead to variable Eimeria oocyst viability, which is not readily assessed microscopically. Under fluorescent microscopy, some aged Eimeria maxima (EM) oocysts were strongly autofluorescent (AF) within the oocystoplasm and sporocysts, whereas others were distinctly non-fluorescent, leading to the hypothesis that non-viable oocysts may be detectible using this simple approach. Using accelerated aging conditions at 45°C, two experiments were conducted to evaluate variable percentages of autofluorescent EM oocysts on body weight gain (BWG), lesion scores (LS), and total oocyst shedding (OS) per bird. Through oral gavage, EM oocysts were administered on d10, whereas LS and BWG were determined d5 post-inoculation. Experiment 1 groups consisted of non-challenged controls (n = 15), or 25,000 EM exhibiting 12.8% (n = 14), 61.1% (n = 10), or 93.3% (n = 10) autofluorescence. BWG in 12.8% AF group was lower (P = 0.054) than non-challenged control. LS were higher (P < 0.05) in 61.1% and 12.8% AF groups as compared to non-challenged control and 93.3% AF groups. Experiment 2 groups consisted of non-challenged controls (LS n = 20/BWG n = 40), or 22,500 EM exhibiting 7% AF (LS n = 20/BWG n = 40), 80.6% AF (LS n = 19/BWG n = 39), or 99% AF (LS n = 19/BWG n = 39). BWG in 7% AF group was lower (P < 0.05) than non-challenged control and 99% AF groups. LS were higher (P < 0.05) in 80.6% and 7% AF groups as compared to non-challenged control and 99% AF groups. OS from d5–8 post-inoculation was determined for each of five replicates per group (n = 20/group; n = 4/replicate), with higher (P < 0.05) OS in 80.6% and 7% AF groups than in non-challenged control or 99% AF groups. Taken together, data indicate lower LS, higher BWG, and reduced OS with higher %AF oocysts, consistent with the hypothesis of lowered viable challenge with this EM isolate. INTRODUCTION Coccidiosis of poultry, caused by Eimeria spp., is one of the top problems facing the poultry industry (Shivaramaiah et al., 2014; Swaggerty et al., 2015; Price et al., 2016). Global economic losses from coccidiosis exceed $800 million USD in the United States and $3 billion USD worldwide (Sharman et al., 2010; Shivaramaiah et al., 2014; Swaggerty et al., 2015). Of those costs, at least 70% results from lower than expected flock performance, which includes reduced weight gain and poor feed conversion—characteristics of subclinical coccidiosis (Swaggerty et al., 2015). Recent legislative and social pressures to remove traditional anticoccidial ionophores from inclusion in feed have left large sectors of the poultry industry with the single option of vaccination of chicks at hatch, either as a sole method for control, or intermittently to refresh resident population sensitivity to chemical coccidiostats (Williams, 2002; Cervantes, 2015). Vaccination at hatch relies on a controlled early exposure to low-level sporulated and infective oocysts, so that chicks develop immunity as these important parasites cycle during the first few weeks of life. Among other factors, the dosage of viable Eimeria oocysts is critical for effective vaccination without causing overt disease, because, with the exception of precocious strains (Williams, 2002; Chapman, 2014; Shivaramaiah et al., 2014), these are generally fully virulent strains (Shivaramaiah et al., 2014). Therefore, a major problem facing both the vaccine and poultry industries is the current inability to discretely enumerate viable oocysts for consistent vaccine or experimental challenge performance (Jenkins et al., 2013). Depending on storage conditions and duration of holding, it is well documented that oocyst preparations lose viability and become non-infective (Augustine, 1980). Recently, a high-fidelity fluorescent microscopy approach with DIC images overlaid with fluorescent images (Figure 1) showed some oocysts with microscopic morphology consistent with mature sporulated and viable oocysts that exhibited strong autofluorescence within the oocystoplasm and sporocysts, whereas others were distinctly devoid of such AF (Hargis et al., unpublished data). In these initial observations, older Eimeria maxima (Guelph strain) batches (held for months at 4°C) had a much higher incidence of autofluorescent (AF) oocysts with normal sporulated morphology under DIC microscopy, suggesting that aging was associated with increased incidence of AF, leading to the hypothesis that this might be an indicator of non-viability of individual oocysts. The present manuscript describes a preliminary attempt to evaluate this hypothesis in a single Eimeria species under accelerated aging at 45°C. Figure 1. View largeDownload slide Left panel—Conventional DIC photomicrograph showing 10 mature Eimeria maxima oocysts. All but one (unsporulated, indicated by asterisk) of the oocysts on this field would be counted as potentially viable oocysts using conventional counting for vaccine enumeration. Nevertheless, in this aged oocyst preparation, we expect that a significant percentage are non-viable. An incidental observation in our laboratory indicated that 4 of these 10 oocysts fluoresced brightly under high-intensity fluorescent microscopy (right panel). An example of what we hypothesized is a viable non-fluorescent mature oocyst as indicated by the arrow (1), whereas a hypothetical non-viable oocyst is indicated by the arrow (2). Figure 1. View largeDownload slide Left panel—Conventional DIC photomicrograph showing 10 mature Eimeria maxima oocysts. All but one (unsporulated, indicated by asterisk) of the oocysts on this field would be counted as potentially viable oocysts using conventional counting for vaccine enumeration. Nevertheless, in this aged oocyst preparation, we expect that a significant percentage are non-viable. An incidental observation in our laboratory indicated that 4 of these 10 oocysts fluoresced brightly under high-intensity fluorescent microscopy (right panel). An example of what we hypothesized is a viable non-fluorescent mature oocyst as indicated by the arrow (1), whereas a hypothetical non-viable oocyst is indicated by the arrow (2). MATERIALS AND METHODS Experiment 1 Experimental Birds. A total of 49 1-d-old broiler chicks were individually neck-tagged and randomly allocated to floor pens at The Ohio Agricultural Research and Development Center Chicken Research Farm of The Ohio State University (Wooster). All procedures followed Institutional Animal Care and Use Committee (IACUC protocol #2015A00000106). Feed met or exceeded NRC requirements (NRC, 1994). Chicks had ad libitum access to feed and water throughout the experiment. Eimeria Maxima. Eimeria maxima (Guelph strain) oocysts were held at 4°C. A subpopulation was placed in an incubator to age for 6d at 45°C, a process known to accelerate aging and reduce viability (Augustine, 1980). All chicks except the negative controls were individually challenged at d10 with 25,000 fresh (maintained at 4°C), aged (6d at 45°C), or a ratio of aged/fresh sporulated oocysts of E. maxima through oral gavage. The fresh, aged, and ratio of aged/fresh oocysts contained 12.8%, 93.3%, and 61.1% AF sporulated oocysts, respectively, at the time of inoculation. Autofluorescence was determined using a Zeiss Axio Imager M2 430004-9902. Images for enumeration of %AF were obtained using the Zeiss Imager M2 with a 20× Plan-APOCHROMAT 20×/0.8 objective and a 100× EC PLAN-NEOFLUOR 100×/1.3 oil objective. The AF was observed through filter set 38 1031-346 with an excitation of BP 470/40, beamsplitter of FT 495, and emission spectrum of BP 525/50. Differential interference contrast images were collected using DIC M27 condensers. The AF was excited for 1 s prior to capturing each image using an Axio Cam MR3 camera. All analysis was performed using AxioVision SE64 4.9.1 SP1 software (Carl Zeiss Microscopy GmbH 2006–2013, Jena, Germany). Lesion Scores and BWG. At d10 and d15, all chicks were individually weighed for calculation of post-inoculation BWG. All chicks (negative control n = 15; aged (93.3%AF) n = 10; aged/fresh ratio (61.1%AF) n = 10; fresh (12.8%AF) n = 14) were euthanized d5 post-inoculation (d15 of age) by cervical dislocation under approved IACUC protocol. Gross (macroscopic) lesion scores (LS) were recorded according to previously described methods (Johnson and Reid, 1970). The individual estimating the LS was unaware of treatment groups. Statistical Analysis. Data were computed using JMP Pro 12 software. Significant differences between BWG in treatment groups were determined utilizing ANOVA. When appropriate, means were separated using Tukey's multiple comparison post hoc test with values of P < 0.05 considered significant. Gross LS data were analyzed using the Wilcoxon non-parametric comparisons method where LS were placed into a positive or negative binary list in order to separate for chi-square analysis. Lesion scores of 1 through 4 were assigned to the positive column with values of 1, whereas LS of 0 were assigned to the negative column with values of 0. Experiment 2 Experimental Birds. A total of 160 1-d-old broiler byproduct male chicks were obtained from a local commercial hatchery. Chicks were individually neck-tagged and randomly allocated to four wire battery cages (one battery per treatment group) at the University of Arkansas Poultry Health Laboratory. Two chicks died after placement from issues unrelated to experimental treatments. Wire battery cages were 24 × 33 inches (792 square inches). Each of the four battery cages had two sections of n = 20 chicks each until d10. On d10, each battery was divided into seven sections (five replicates, n = 4 birds for oocyst shedding (OS); two replicates, n = 10 birds for LS). All animal-handling procedures were in compliance with the Institutional Animal Care and Use Committee (IACUC protocol #17017) of the University of Arkansas. A corn-soy-based starter feed that met or exceeded nutrient requirements of poultry (NRC, 1994) and water were provided ad libitum. Clinacox (a commercially available anticoccidial) was included at 1 lb/ton for the first 7 d, and then replaced with non-medicated feed for the duration of the experiment. This was to reduce possibility of unintentional introduction of these ubiquitous parasites prior to challenge on d10. Eimeria Maxima. Eimeria maxima (Guelph Strain) oocysts were held at 4°C, and a subpopulation was placed in an incubator to age for 6d at 45°C as described above. Dosage was based upon a pre-challenge study titrated to reduce BWG during challenge period by approximately 24%. All chicks other than those in the negative control group were individually challenged at d10 with 22,500 fresh (maintained at 4°C), aged (6d at 45°C), or a ratio of aged/fresh sporulated oocysts of E. maxima through oral gavage. The fresh, aged, and ratio of aged/fresh oocysts contained 7%, 99%, and 80.6% AF sporulated oocysts, respectively, at the time of inoculation. Autofluorescence was determined as described above. Enumeration. Mature sporulated E. maxima oocysts, containing sporocysts, were enumerated from excreta samples according to a modified McMaster chamber method (Holdsworth et al., 2004). Ten and 100-fold dilutions of shaken excreta slurry in saturated salt float solution were made. This excreta + saturated salt slurry was then inverted gently to homogenize the suspension without causing air bubbles. Approximately 600 μL was then pipetted into the first McMaster Chamber. The tube was recapped and inverted to redistribute oocysts prior to pipetting of the slurry into the second chamber. The McMaster chamber was then allowed to set for 2–5 min prior to examination to allow floatation of oocysts. Total numbers of oocysts within the marked grids were enumerated. The numbers of oocysts per gram of excreta content were calculated by the following equation: \begin{eqnarray*} {[ {\left( {Chamber\ 1 + Chamber\ 2} \right)}} \nonumber\\ \times \left( {Potassium\,dichromate\,dilution\,factor} \right)\nonumber\\ \times \left( {Saturated\,salt\,dilution\,factor} \right) \nonumber\\ \times \left( {0.3\ \,mL/Chamber} \right) ]. \end{eqnarray*} Lesion Scores and BWG. At d10 and d15, all chicks were individually weighed for calculation of post-inoculation BWG. Twenty chicks from each treatment group (80 total chicks) were euthanized on d5 post-inoculation by cervical dislocation. Gross LS were recorded according to previously described methods (Johnson and Reid, 1970). Oocyst Collection. At d15–18 (d5–8 post-inoculation), all droppings were collected from each replicate within each of the four wire battery cages (five replicates, n = 4 birds for OS; n = 20 total per battery). All excreta from each replicate were collected and stored in separate containers in order to differentiate replicate counts to quantify total OS utilizing a modified McMaster technique as previously described (Hodgson, 1970; Long et al., 1976). Briefly, sheets of freezer paper lined the dropping pans within each replicate for excreta collection. Excreta collection occurred four times per day (approximately 6:00 am, 12:00 pm, 6:00 pm, and 10:00 pm). Wooden craft sticks were utilized to scrape all excreta from the dropping pans and paper beneath each replicate into pre-weighed jars containing potassium dichromate (1.5% concentration) at determined volume no less than 2 parts excreta to 1 part potassium dichromate. Wooden craft sticks were changed between replicates and treatment groups to prevent contamination. Fresh sheets of freezer paper were placed in the dropping pans following each collection. Additionally, gloves were changed between treatment groups to prevent contamination. Following each collection, the experiment room walls, floors, and other exposed areas were sprayed with water to maintain humidity to prevent excreta drying, thus preserving oocyst integrity. Minimum fresh air ventilation was provided in order to help prevent further drying of excreta. After collection, samples were stored at 4°C for inhibition of bacterial growth. Initial total weight of the jar and potassium dichromate was subtracted from the final jar weight (potassium dichromate, jar, and excreta) to determine the total grams of excreta collected. Due to the high volume of excreta samples, multiple excreta sample jars were utilized for each replicate. Therefore, following the final d18 (d8 post-inoculation) collection, samples from respective replicates were homogenized, and then combined into one pooled sample for that respective replicate, resulting in n = 20 total OS count samples (four groups of five replicates). Statistical Analysis. Data were analyzed using JMP Pro 12 software, with significant differences between OS and BWG among treatment groups analyzed using ANOVA as described above. Factors included treatment group of inoculated oocyst dose and BWG. Lesion scores were analyzed using the Wilcoxon non-parametric comparisons method as described above. RESULTS AND DISCUSSION Avian Eimeria Infection Response and Lesions Initially, an increase in oocyst dosage results in progressively increased oocyst yields; however, this only continues until the “maximally producing dose”, whereby increased dosage actually yields progressively decreasing oocyst numbers (Williams, 2001). “Crowded threshold” marks the point whereby increase in dosage no longer yields increased oocyst numbers, whereas “crowded doses” refers to the dosage exceeding that amount (Williams, 2001). Parasite fecundity has been shown to decrease as the infectious dose of Eimeria spp. increases (Johnston et al., 2001; Williams, 2001). This “crowding effect” can especially be observed in naïve hosts that have been challenged with sporulated oocysts (Williams, 2001). Specific lesions and location depend upon the species of Eimeria, but a primary response within the intestinal tissue following coccidial infection is edema within the submucosal layer (Long et al., 1976). Invasion of the villi results in sloughing of epithelial cells, shortening of villi, and flattening of the mucosa (Long et al., 1976). Hence, the incidence of enteric diseases such as coccidiosis continues to be an expensive problem within the poultry industry (Shivaramaiah et al., 2014). Within the present studies using the Guelph strain of E. maxima, modest ballooning and thickening of the intestinal wall were observed, and the intestinal tract was bleached moderately. The serosal surface had diffuse red petechiae and the mucosal surface was covered partially by orange mucus. The region from below the duodenal loop through to Meckel's diverticulum was affected with the most severe lesions observed near the midpoint of this affected region. Autofluorescence Length of storage time and temperature has been shown to negatively impact viability of coccidian oocysts (Augustine, 1980). In a study completed by Augustine (1980), E. meleagrimitis oocysts stored at different temperatures for up to one year exhibited decreasing amylopectin levels (glucose measured) and viability (oocyst numbers and overall turkey mortality) in relationship to storage length and temperature. Amylopectin is considered a polysaccharide energy source for the sporulation process of Eimeria oocysts, and, later, for survival of sporozoites (Augustine, 1980). Within the sexual stage of E. maxima, gam56 and gam82, two tyrosine-rich precursor glycoproteins, are found within the wall-forming bodies that are special organelles within the oocyst (Belli et al., 2003). Through proteolytic processing, these glycoproteins are broken into smaller glycoproteins which are then incorporated into oocyst wall development (Belli et al., 2003; Belli et al., 2006; Chapman, 2014). High concentrations of dityrosine and 3,4-dihydroxyphenylalanine (DOPA) have been identified within oocyst extracts through methods of high-pressure liquid chromatography. These concentrations as well as UV autofluorescence detection in intact oocysts associate the possible role that dityrosine and DOPA–protein cross-links play in the hardening of oocyst walls (Belli et al., 2003). Belli et al. (2003) conducted a study with Eimeria spp. to show that presence of dityrosine cross-links was associated with AF in oocysts. Even without an understanding of the molecular fluorescence properties, Eimeria AF has been used diagnostically in the identification of Eimeria oocyst species in both fish tissues and swine excreta matter (Belli et al., 2003). Under UV illumination and view with correct UV excitation and filter set, coccidian oocysts have been shown to autofluoresce blue, allowing identification even from complex microscopic backgrounds (Lindquist et al., 2003; Belli et al., 2006). Belli et al. (2003, 2006) provided evidence of dityrosine cross-links as a causative agent of natural AF within Eimeria oocysts. However, to our knowledge, no investigations have reported any association of AF and Eimeria viability. The present work describes the association of AF E. maxima oocysts and viability through evaluation of LS, OS, and BWG during 5–8 days post-inoculation. We further speculate that this approach could lead to more consistent enumeration of viable Eimeria for vaccine production or experimental challenge studies. If an economical measure of coccidian viability were produced, it could potentially markedly improve the ability to titrate coccidiosis vaccines and improve reproducibility of coccidiosis research. Experiment 1 No statistical difference was detected in post-challenge BWG (Table 1); although when a one-way ANOVA was analyzed between the negative control and fresh (12.8% AF) treatment groups, these two groups were different (P = 0.054). However, post-challenge BWG was shown to generally decrease as %AF decreased, indicating a possible greater disease challenge and viability within oocysts that are not AF. The ratio of aged/fresh (61.1% AF) and fresh (12.8% AF) as compared to negative control and aged (93.3% AF) challenge groups was statistically different (P < 0.05) in LS. This response was consistent with our hypothesis of AF being related to non-viability. The fresh (12.8% AF) and ratio of aged/fresh (61.1% AF) groups appeared to suffer greater disease challenge as indicated by decreased post-challenge BWG and increased LS as compared to the negative control and aged (93.3% AF). Table 1. Effect of challenge with fresh (maintained at 4°C), aged (6 d at 45°C), or a ratio of aged/fresh Eimeria maxima oocysts at 10 days of age on d15 lesion scores and d10–d15 BWG as compared to non-challenged controls (Experiment 1). Eimeria maxima Challenge level %AF1 Post-challenge BWG2 (g) Gross lesion score3 Negative control N/A 225 ± 10.8 0.21 ± 0.11b 25,000 Aged 93.3 228 ± 6.9 0.30 ± 0.15b 25,000 Aged:fresh (60%:40%) 61.1 212 ± 10.6 1.20 ± 0.13a 25,000 Fresh 12.8 206 ± 5.33 0.86 ± 0.14a Eimeria maxima Challenge level %AF1 Post-challenge BWG2 (g) Gross lesion score3 Negative control N/A 225 ± 10.8 0.21 ± 0.11b 25,000 Aged 93.3 228 ± 6.9 0.30 ± 0.15b 25,000 Aged:fresh (60%:40%) 61.1 212 ± 10.6 1.20 ± 0.13a 25,000 Fresh 12.8 206 ± 5.33 0.86 ± 0.14a a,bMeans ± SEM within variable columns with different superscripts are significantly different (P < 0.05). 1AF = Autofluorescence. Determined using Zeiss Axio Imager M2 430004-9902 with filter set 38 1031-346 with an excitation of BP 470/40, beamsplitter of FT 495, and emission spectrum of BP 525/50. 2Although there were no significant differences, the negative control was compared to the 25,000 fresh using a one-way ANOVA (P = 0.054). 3Analyzed using non-parametric comparisons Wilcoxon method. View Large Table 1. Effect of challenge with fresh (maintained at 4°C), aged (6 d at 45°C), or a ratio of aged/fresh Eimeria maxima oocysts at 10 days of age on d15 lesion scores and d10–d15 BWG as compared to non-challenged controls (Experiment 1). Eimeria maxima Challenge level %AF1 Post-challenge BWG2 (g) Gross lesion score3 Negative control N/A 225 ± 10.8 0.21 ± 0.11b 25,000 Aged 93.3 228 ± 6.9 0.30 ± 0.15b 25,000 Aged:fresh (60%:40%) 61.1 212 ± 10.6 1.20 ± 0.13a 25,000 Fresh 12.8 206 ± 5.33 0.86 ± 0.14a Eimeria maxima Challenge level %AF1 Post-challenge BWG2 (g) Gross lesion score3 Negative control N/A 225 ± 10.8 0.21 ± 0.11b 25,000 Aged 93.3 228 ± 6.9 0.30 ± 0.15b 25,000 Aged:fresh (60%:40%) 61.1 212 ± 10.6 1.20 ± 0.13a 25,000 Fresh 12.8 206 ± 5.33 0.86 ± 0.14a a,bMeans ± SEM within variable columns with different superscripts are significantly different (P < 0.05). 1AF = Autofluorescence. Determined using Zeiss Axio Imager M2 430004-9902 with filter set 38 1031-346 with an excitation of BP 470/40, beamsplitter of FT 495, and emission spectrum of BP 525/50. 2Although there were no significant differences, the negative control was compared to the 25,000 fresh using a one-way ANOVA (P = 0.054). 3Analyzed using non-parametric comparisons Wilcoxon method. View Large Experiment 2 Utilizing ANOVA, the fresh (7% AF) treatment group was detected as statistically different (P < 0.05) in post-challenge BWG when compared to the negative control and aged (99% AF) groups (Table 2). The ratio aged/fresh (80.6% AF) treatment group was determined to be statistically different (P < 0.05) from the negative control in post-challenge BWG. Both the ratio aged/fresh (80.6%AF) and fresh (7% AF) treatment groups were statistically different from the negative control and aged (99% AF) groups in LS. Table 2. Effect of challenge with fresh (maintained at 4°C), aged (6d at 45°C), or a ratio of aged:fresh Eimeria maxima oocysts at 10 days of age on d15 lesion scores, d10–d15 BWG, and mean total oocyst shedding from d15–18 per bird as compared to non-challenged controls (Experiment 2). Eimeria maxima Challenge level %AF1 Post-challenge BWG2 (g) Gross lesion score3 Mean total OPG/bird2 Negative control N/A 226 ± 4.95a 0.00 ± 0.00b 0 ± 0.00b 22,500 Aged 99 214 ± 7.72a,b 0.11 ± 0.11b 0 ± 0.00b 22,500 Aged:fresh (80%:20%) 80.6 197 ± 9.14b,c 1.42 ± 0.14a 24,569 ± 1,903a 22,500 Fresh 7 176 ± 6.25c 2.00 ± 0.10a 24,964 ± 2,334a Eimeria maxima Challenge level %AF1 Post-challenge BWG2 (g) Gross lesion score3 Mean total OPG/bird2 Negative control N/A 226 ± 4.95a 0.00 ± 0.00b 0 ± 0.00b 22,500 Aged 99 214 ± 7.72a,b 0.11 ± 0.11b 0 ± 0.00b 22,500 Aged:fresh (80%:20%) 80.6 197 ± 9.14b,c 1.42 ± 0.14a 24,569 ± 1,903a 22,500 Fresh 7 176 ± 6.25c 2.00 ± 0.10a 24,964 ± 2,334a a–cMeans ± SEM within variable columns with different superscripts are significantly different (P < 0.05). 1AF = Autofluorescence. Determined using Zeiss Axio Imager M2 430004-9902 with filter set 38 1031-346 with an excitation of BP 470/40, beamsplitter of FT 495, and emission spectrum of BP 525/50. 2Statistical evaluation using ANOVA followed by Tukey's multiple comparison post hoc test. 3Analyzed using non-parametric comparisons Wilcoxon method. View Large Table 2. Effect of challenge with fresh (maintained at 4°C), aged (6d at 45°C), or a ratio of aged:fresh Eimeria maxima oocysts at 10 days of age on d15 lesion scores, d10–d15 BWG, and mean total oocyst shedding from d15–18 per bird as compared to non-challenged controls (Experiment 2). Eimeria maxima Challenge level %AF1 Post-challenge BWG2 (g) Gross lesion score3 Mean total OPG/bird2 Negative control N/A 226 ± 4.95a 0.00 ± 0.00b 0 ± 0.00b 22,500 Aged 99 214 ± 7.72a,b 0.11 ± 0.11b 0 ± 0.00b 22,500 Aged:fresh (80%:20%) 80.6 197 ± 9.14b,c 1.42 ± 0.14a 24,569 ± 1,903a 22,500 Fresh 7 176 ± 6.25c 2.00 ± 0.10a 24,964 ± 2,334a Eimeria maxima Challenge level %AF1 Post-challenge BWG2 (g) Gross lesion score3 Mean total OPG/bird2 Negative control N/A 226 ± 4.95a 0.00 ± 0.00b 0 ± 0.00b 22,500 Aged 99 214 ± 7.72a,b 0.11 ± 0.11b 0 ± 0.00b 22,500 Aged:fresh (80%:20%) 80.6 197 ± 9.14b,c 1.42 ± 0.14a 24,569 ± 1,903a 22,500 Fresh 7 176 ± 6.25c 2.00 ± 0.10a 24,964 ± 2,334a a–cMeans ± SEM within variable columns with different superscripts are significantly different (P < 0.05). 1AF = Autofluorescence. Determined using Zeiss Axio Imager M2 430004-9902 with filter set 38 1031-346 with an excitation of BP 470/40, beamsplitter of FT 495, and emission spectrum of BP 525/50. 2Statistical evaluation using ANOVA followed by Tukey's multiple comparison post hoc test. 3Analyzed using non-parametric comparisons Wilcoxon method. View Large There was no detectible OS within the negative control and aged (99%AF) treatment groups, whereas significant numbers were detected within the ratio aged/fresh (80.6% AF) and fresh (7%AF) treatment groups (Table 2). The ratio aged/fresh (80.6% AF) and fresh (7% AF) treatment groups were found to have OS significantly different from the negative control and aged (99% AF) treatment groups. However, the lack of statistical difference between the ratio (80.6% AF) and fresh (7% AF) groups can potentially be explained by “crowding effect” as mentioned earlier (Johnston et al., 2001; Williams, 2001). Overall, it appeared that increased %AF was consistent with our hypothesis of non-viability within Eimeria oocysts. The treatment groups with higher %AF resulted in a general consistency of lower LS, higher BWG, and reduced OS as compared to the negative control and aged oocyst treatment groups—suggesting lowered disease challenge. This may suggest that AF could be a non-viability indicator, potentially offering an alternative method for easy enumeration for vaccine titration or research challenge studies. However, more research is needed among the various Eimeria species to determine whether AF is a consistent indicator of non-viability for all species. Moreover, although AF in fresh and aged oocysts has been associated with increased levels of dityrosine crosslinks (Belli et al., 2003, 2006), the AF substance(s) associated with accelerated aging in the present studies were not identified. REFERENCES Augustine P. C. 1980 . 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Quantification of the crowding effect during infections with the seven Eimeria species of the domesticated fowl: its importance for experimental designs and the production of oocyst stocks . Int. J. Parasitol. 31 : 1056 – 1069 . Google Scholar CrossRef Search ADS PubMed Williams R. B. 2002 . Anticoccidial vaccines for broiler chickens: pathways to success . Avian Pathol. 31 : 317 – 353 . Google Scholar CrossRef Search ADS PubMed © 2018 Poultry Science Association Inc. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Poultry Science Oxford University Press

Evaluation of autofluorescent Eimeria maxima oocysts as a potential indicator of non-viability when enumerating oocysts

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© 2018 Poultry Science Association Inc.
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Abstract

Abstract Aging, poor oxygenation, or improper storage temperature can lead to variable Eimeria oocyst viability, which is not readily assessed microscopically. Under fluorescent microscopy, some aged Eimeria maxima (EM) oocysts were strongly autofluorescent (AF) within the oocystoplasm and sporocysts, whereas others were distinctly non-fluorescent, leading to the hypothesis that non-viable oocysts may be detectible using this simple approach. Using accelerated aging conditions at 45°C, two experiments were conducted to evaluate variable percentages of autofluorescent EM oocysts on body weight gain (BWG), lesion scores (LS), and total oocyst shedding (OS) per bird. Through oral gavage, EM oocysts were administered on d10, whereas LS and BWG were determined d5 post-inoculation. Experiment 1 groups consisted of non-challenged controls (n = 15), or 25,000 EM exhibiting 12.8% (n = 14), 61.1% (n = 10), or 93.3% (n = 10) autofluorescence. BWG in 12.8% AF group was lower (P = 0.054) than non-challenged control. LS were higher (P < 0.05) in 61.1% and 12.8% AF groups as compared to non-challenged control and 93.3% AF groups. Experiment 2 groups consisted of non-challenged controls (LS n = 20/BWG n = 40), or 22,500 EM exhibiting 7% AF (LS n = 20/BWG n = 40), 80.6% AF (LS n = 19/BWG n = 39), or 99% AF (LS n = 19/BWG n = 39). BWG in 7% AF group was lower (P < 0.05) than non-challenged control and 99% AF groups. LS were higher (P < 0.05) in 80.6% and 7% AF groups as compared to non-challenged control and 99% AF groups. OS from d5–8 post-inoculation was determined for each of five replicates per group (n = 20/group; n = 4/replicate), with higher (P < 0.05) OS in 80.6% and 7% AF groups than in non-challenged control or 99% AF groups. Taken together, data indicate lower LS, higher BWG, and reduced OS with higher %AF oocysts, consistent with the hypothesis of lowered viable challenge with this EM isolate. INTRODUCTION Coccidiosis of poultry, caused by Eimeria spp., is one of the top problems facing the poultry industry (Shivaramaiah et al., 2014; Swaggerty et al., 2015; Price et al., 2016). Global economic losses from coccidiosis exceed $800 million USD in the United States and $3 billion USD worldwide (Sharman et al., 2010; Shivaramaiah et al., 2014; Swaggerty et al., 2015). Of those costs, at least 70% results from lower than expected flock performance, which includes reduced weight gain and poor feed conversion—characteristics of subclinical coccidiosis (Swaggerty et al., 2015). Recent legislative and social pressures to remove traditional anticoccidial ionophores from inclusion in feed have left large sectors of the poultry industry with the single option of vaccination of chicks at hatch, either as a sole method for control, or intermittently to refresh resident population sensitivity to chemical coccidiostats (Williams, 2002; Cervantes, 2015). Vaccination at hatch relies on a controlled early exposure to low-level sporulated and infective oocysts, so that chicks develop immunity as these important parasites cycle during the first few weeks of life. Among other factors, the dosage of viable Eimeria oocysts is critical for effective vaccination without causing overt disease, because, with the exception of precocious strains (Williams, 2002; Chapman, 2014; Shivaramaiah et al., 2014), these are generally fully virulent strains (Shivaramaiah et al., 2014). Therefore, a major problem facing both the vaccine and poultry industries is the current inability to discretely enumerate viable oocysts for consistent vaccine or experimental challenge performance (Jenkins et al., 2013). Depending on storage conditions and duration of holding, it is well documented that oocyst preparations lose viability and become non-infective (Augustine, 1980). Recently, a high-fidelity fluorescent microscopy approach with DIC images overlaid with fluorescent images (Figure 1) showed some oocysts with microscopic morphology consistent with mature sporulated and viable oocysts that exhibited strong autofluorescence within the oocystoplasm and sporocysts, whereas others were distinctly devoid of such AF (Hargis et al., unpublished data). In these initial observations, older Eimeria maxima (Guelph strain) batches (held for months at 4°C) had a much higher incidence of autofluorescent (AF) oocysts with normal sporulated morphology under DIC microscopy, suggesting that aging was associated with increased incidence of AF, leading to the hypothesis that this might be an indicator of non-viability of individual oocysts. The present manuscript describes a preliminary attempt to evaluate this hypothesis in a single Eimeria species under accelerated aging at 45°C. Figure 1. View largeDownload slide Left panel—Conventional DIC photomicrograph showing 10 mature Eimeria maxima oocysts. All but one (unsporulated, indicated by asterisk) of the oocysts on this field would be counted as potentially viable oocysts using conventional counting for vaccine enumeration. Nevertheless, in this aged oocyst preparation, we expect that a significant percentage are non-viable. An incidental observation in our laboratory indicated that 4 of these 10 oocysts fluoresced brightly under high-intensity fluorescent microscopy (right panel). An example of what we hypothesized is a viable non-fluorescent mature oocyst as indicated by the arrow (1), whereas a hypothetical non-viable oocyst is indicated by the arrow (2). Figure 1. View largeDownload slide Left panel—Conventional DIC photomicrograph showing 10 mature Eimeria maxima oocysts. All but one (unsporulated, indicated by asterisk) of the oocysts on this field would be counted as potentially viable oocysts using conventional counting for vaccine enumeration. Nevertheless, in this aged oocyst preparation, we expect that a significant percentage are non-viable. An incidental observation in our laboratory indicated that 4 of these 10 oocysts fluoresced brightly under high-intensity fluorescent microscopy (right panel). An example of what we hypothesized is a viable non-fluorescent mature oocyst as indicated by the arrow (1), whereas a hypothetical non-viable oocyst is indicated by the arrow (2). MATERIALS AND METHODS Experiment 1 Experimental Birds. A total of 49 1-d-old broiler chicks were individually neck-tagged and randomly allocated to floor pens at The Ohio Agricultural Research and Development Center Chicken Research Farm of The Ohio State University (Wooster). All procedures followed Institutional Animal Care and Use Committee (IACUC protocol #2015A00000106). Feed met or exceeded NRC requirements (NRC, 1994). Chicks had ad libitum access to feed and water throughout the experiment. Eimeria Maxima. Eimeria maxima (Guelph strain) oocysts were held at 4°C. A subpopulation was placed in an incubator to age for 6d at 45°C, a process known to accelerate aging and reduce viability (Augustine, 1980). All chicks except the negative controls were individually challenged at d10 with 25,000 fresh (maintained at 4°C), aged (6d at 45°C), or a ratio of aged/fresh sporulated oocysts of E. maxima through oral gavage. The fresh, aged, and ratio of aged/fresh oocysts contained 12.8%, 93.3%, and 61.1% AF sporulated oocysts, respectively, at the time of inoculation. Autofluorescence was determined using a Zeiss Axio Imager M2 430004-9902. Images for enumeration of %AF were obtained using the Zeiss Imager M2 with a 20× Plan-APOCHROMAT 20×/0.8 objective and a 100× EC PLAN-NEOFLUOR 100×/1.3 oil objective. The AF was observed through filter set 38 1031-346 with an excitation of BP 470/40, beamsplitter of FT 495, and emission spectrum of BP 525/50. Differential interference contrast images were collected using DIC M27 condensers. The AF was excited for 1 s prior to capturing each image using an Axio Cam MR3 camera. All analysis was performed using AxioVision SE64 4.9.1 SP1 software (Carl Zeiss Microscopy GmbH 2006–2013, Jena, Germany). Lesion Scores and BWG. At d10 and d15, all chicks were individually weighed for calculation of post-inoculation BWG. All chicks (negative control n = 15; aged (93.3%AF) n = 10; aged/fresh ratio (61.1%AF) n = 10; fresh (12.8%AF) n = 14) were euthanized d5 post-inoculation (d15 of age) by cervical dislocation under approved IACUC protocol. Gross (macroscopic) lesion scores (LS) were recorded according to previously described methods (Johnson and Reid, 1970). The individual estimating the LS was unaware of treatment groups. Statistical Analysis. Data were computed using JMP Pro 12 software. Significant differences between BWG in treatment groups were determined utilizing ANOVA. When appropriate, means were separated using Tukey's multiple comparison post hoc test with values of P < 0.05 considered significant. Gross LS data were analyzed using the Wilcoxon non-parametric comparisons method where LS were placed into a positive or negative binary list in order to separate for chi-square analysis. Lesion scores of 1 through 4 were assigned to the positive column with values of 1, whereas LS of 0 were assigned to the negative column with values of 0. Experiment 2 Experimental Birds. A total of 160 1-d-old broiler byproduct male chicks were obtained from a local commercial hatchery. Chicks were individually neck-tagged and randomly allocated to four wire battery cages (one battery per treatment group) at the University of Arkansas Poultry Health Laboratory. Two chicks died after placement from issues unrelated to experimental treatments. Wire battery cages were 24 × 33 inches (792 square inches). Each of the four battery cages had two sections of n = 20 chicks each until d10. On d10, each battery was divided into seven sections (five replicates, n = 4 birds for oocyst shedding (OS); two replicates, n = 10 birds for LS). All animal-handling procedures were in compliance with the Institutional Animal Care and Use Committee (IACUC protocol #17017) of the University of Arkansas. A corn-soy-based starter feed that met or exceeded nutrient requirements of poultry (NRC, 1994) and water were provided ad libitum. Clinacox (a commercially available anticoccidial) was included at 1 lb/ton for the first 7 d, and then replaced with non-medicated feed for the duration of the experiment. This was to reduce possibility of unintentional introduction of these ubiquitous parasites prior to challenge on d10. Eimeria Maxima. Eimeria maxima (Guelph Strain) oocysts were held at 4°C, and a subpopulation was placed in an incubator to age for 6d at 45°C as described above. Dosage was based upon a pre-challenge study titrated to reduce BWG during challenge period by approximately 24%. All chicks other than those in the negative control group were individually challenged at d10 with 22,500 fresh (maintained at 4°C), aged (6d at 45°C), or a ratio of aged/fresh sporulated oocysts of E. maxima through oral gavage. The fresh, aged, and ratio of aged/fresh oocysts contained 7%, 99%, and 80.6% AF sporulated oocysts, respectively, at the time of inoculation. Autofluorescence was determined as described above. Enumeration. Mature sporulated E. maxima oocysts, containing sporocysts, were enumerated from excreta samples according to a modified McMaster chamber method (Holdsworth et al., 2004). Ten and 100-fold dilutions of shaken excreta slurry in saturated salt float solution were made. This excreta + saturated salt slurry was then inverted gently to homogenize the suspension without causing air bubbles. Approximately 600 μL was then pipetted into the first McMaster Chamber. The tube was recapped and inverted to redistribute oocysts prior to pipetting of the slurry into the second chamber. The McMaster chamber was then allowed to set for 2–5 min prior to examination to allow floatation of oocysts. Total numbers of oocysts within the marked grids were enumerated. The numbers of oocysts per gram of excreta content were calculated by the following equation: \begin{eqnarray*} {[ {\left( {Chamber\ 1 + Chamber\ 2} \right)}} \nonumber\\ \times \left( {Potassium\,dichromate\,dilution\,factor} \right)\nonumber\\ \times \left( {Saturated\,salt\,dilution\,factor} \right) \nonumber\\ \times \left( {0.3\ \,mL/Chamber} \right) ]. \end{eqnarray*} Lesion Scores and BWG. At d10 and d15, all chicks were individually weighed for calculation of post-inoculation BWG. Twenty chicks from each treatment group (80 total chicks) were euthanized on d5 post-inoculation by cervical dislocation. Gross LS were recorded according to previously described methods (Johnson and Reid, 1970). Oocyst Collection. At d15–18 (d5–8 post-inoculation), all droppings were collected from each replicate within each of the four wire battery cages (five replicates, n = 4 birds for OS; n = 20 total per battery). All excreta from each replicate were collected and stored in separate containers in order to differentiate replicate counts to quantify total OS utilizing a modified McMaster technique as previously described (Hodgson, 1970; Long et al., 1976). Briefly, sheets of freezer paper lined the dropping pans within each replicate for excreta collection. Excreta collection occurred four times per day (approximately 6:00 am, 12:00 pm, 6:00 pm, and 10:00 pm). Wooden craft sticks were utilized to scrape all excreta from the dropping pans and paper beneath each replicate into pre-weighed jars containing potassium dichromate (1.5% concentration) at determined volume no less than 2 parts excreta to 1 part potassium dichromate. Wooden craft sticks were changed between replicates and treatment groups to prevent contamination. Fresh sheets of freezer paper were placed in the dropping pans following each collection. Additionally, gloves were changed between treatment groups to prevent contamination. Following each collection, the experiment room walls, floors, and other exposed areas were sprayed with water to maintain humidity to prevent excreta drying, thus preserving oocyst integrity. Minimum fresh air ventilation was provided in order to help prevent further drying of excreta. After collection, samples were stored at 4°C for inhibition of bacterial growth. Initial total weight of the jar and potassium dichromate was subtracted from the final jar weight (potassium dichromate, jar, and excreta) to determine the total grams of excreta collected. Due to the high volume of excreta samples, multiple excreta sample jars were utilized for each replicate. Therefore, following the final d18 (d8 post-inoculation) collection, samples from respective replicates were homogenized, and then combined into one pooled sample for that respective replicate, resulting in n = 20 total OS count samples (four groups of five replicates). Statistical Analysis. Data were analyzed using JMP Pro 12 software, with significant differences between OS and BWG among treatment groups analyzed using ANOVA as described above. Factors included treatment group of inoculated oocyst dose and BWG. Lesion scores were analyzed using the Wilcoxon non-parametric comparisons method as described above. RESULTS AND DISCUSSION Avian Eimeria Infection Response and Lesions Initially, an increase in oocyst dosage results in progressively increased oocyst yields; however, this only continues until the “maximally producing dose”, whereby increased dosage actually yields progressively decreasing oocyst numbers (Williams, 2001). “Crowded threshold” marks the point whereby increase in dosage no longer yields increased oocyst numbers, whereas “crowded doses” refers to the dosage exceeding that amount (Williams, 2001). Parasite fecundity has been shown to decrease as the infectious dose of Eimeria spp. increases (Johnston et al., 2001; Williams, 2001). This “crowding effect” can especially be observed in naïve hosts that have been challenged with sporulated oocysts (Williams, 2001). Specific lesions and location depend upon the species of Eimeria, but a primary response within the intestinal tissue following coccidial infection is edema within the submucosal layer (Long et al., 1976). Invasion of the villi results in sloughing of epithelial cells, shortening of villi, and flattening of the mucosa (Long et al., 1976). Hence, the incidence of enteric diseases such as coccidiosis continues to be an expensive problem within the poultry industry (Shivaramaiah et al., 2014). Within the present studies using the Guelph strain of E. maxima, modest ballooning and thickening of the intestinal wall were observed, and the intestinal tract was bleached moderately. The serosal surface had diffuse red petechiae and the mucosal surface was covered partially by orange mucus. The region from below the duodenal loop through to Meckel's diverticulum was affected with the most severe lesions observed near the midpoint of this affected region. Autofluorescence Length of storage time and temperature has been shown to negatively impact viability of coccidian oocysts (Augustine, 1980). In a study completed by Augustine (1980), E. meleagrimitis oocysts stored at different temperatures for up to one year exhibited decreasing amylopectin levels (glucose measured) and viability (oocyst numbers and overall turkey mortality) in relationship to storage length and temperature. Amylopectin is considered a polysaccharide energy source for the sporulation process of Eimeria oocysts, and, later, for survival of sporozoites (Augustine, 1980). Within the sexual stage of E. maxima, gam56 and gam82, two tyrosine-rich precursor glycoproteins, are found within the wall-forming bodies that are special organelles within the oocyst (Belli et al., 2003). Through proteolytic processing, these glycoproteins are broken into smaller glycoproteins which are then incorporated into oocyst wall development (Belli et al., 2003; Belli et al., 2006; Chapman, 2014). High concentrations of dityrosine and 3,4-dihydroxyphenylalanine (DOPA) have been identified within oocyst extracts through methods of high-pressure liquid chromatography. These concentrations as well as UV autofluorescence detection in intact oocysts associate the possible role that dityrosine and DOPA–protein cross-links play in the hardening of oocyst walls (Belli et al., 2003). Belli et al. (2003) conducted a study with Eimeria spp. to show that presence of dityrosine cross-links was associated with AF in oocysts. Even without an understanding of the molecular fluorescence properties, Eimeria AF has been used diagnostically in the identification of Eimeria oocyst species in both fish tissues and swine excreta matter (Belli et al., 2003). Under UV illumination and view with correct UV excitation and filter set, coccidian oocysts have been shown to autofluoresce blue, allowing identification even from complex microscopic backgrounds (Lindquist et al., 2003; Belli et al., 2006). Belli et al. (2003, 2006) provided evidence of dityrosine cross-links as a causative agent of natural AF within Eimeria oocysts. However, to our knowledge, no investigations have reported any association of AF and Eimeria viability. The present work describes the association of AF E. maxima oocysts and viability through evaluation of LS, OS, and BWG during 5–8 days post-inoculation. We further speculate that this approach could lead to more consistent enumeration of viable Eimeria for vaccine production or experimental challenge studies. If an economical measure of coccidian viability were produced, it could potentially markedly improve the ability to titrate coccidiosis vaccines and improve reproducibility of coccidiosis research. Experiment 1 No statistical difference was detected in post-challenge BWG (Table 1); although when a one-way ANOVA was analyzed between the negative control and fresh (12.8% AF) treatment groups, these two groups were different (P = 0.054). However, post-challenge BWG was shown to generally decrease as %AF decreased, indicating a possible greater disease challenge and viability within oocysts that are not AF. The ratio of aged/fresh (61.1% AF) and fresh (12.8% AF) as compared to negative control and aged (93.3% AF) challenge groups was statistically different (P < 0.05) in LS. This response was consistent with our hypothesis of AF being related to non-viability. The fresh (12.8% AF) and ratio of aged/fresh (61.1% AF) groups appeared to suffer greater disease challenge as indicated by decreased post-challenge BWG and increased LS as compared to the negative control and aged (93.3% AF). Table 1. Effect of challenge with fresh (maintained at 4°C), aged (6 d at 45°C), or a ratio of aged/fresh Eimeria maxima oocysts at 10 days of age on d15 lesion scores and d10–d15 BWG as compared to non-challenged controls (Experiment 1). Eimeria maxima Challenge level %AF1 Post-challenge BWG2 (g) Gross lesion score3 Negative control N/A 225 ± 10.8 0.21 ± 0.11b 25,000 Aged 93.3 228 ± 6.9 0.30 ± 0.15b 25,000 Aged:fresh (60%:40%) 61.1 212 ± 10.6 1.20 ± 0.13a 25,000 Fresh 12.8 206 ± 5.33 0.86 ± 0.14a Eimeria maxima Challenge level %AF1 Post-challenge BWG2 (g) Gross lesion score3 Negative control N/A 225 ± 10.8 0.21 ± 0.11b 25,000 Aged 93.3 228 ± 6.9 0.30 ± 0.15b 25,000 Aged:fresh (60%:40%) 61.1 212 ± 10.6 1.20 ± 0.13a 25,000 Fresh 12.8 206 ± 5.33 0.86 ± 0.14a a,bMeans ± SEM within variable columns with different superscripts are significantly different (P < 0.05). 1AF = Autofluorescence. Determined using Zeiss Axio Imager M2 430004-9902 with filter set 38 1031-346 with an excitation of BP 470/40, beamsplitter of FT 495, and emission spectrum of BP 525/50. 2Although there were no significant differences, the negative control was compared to the 25,000 fresh using a one-way ANOVA (P = 0.054). 3Analyzed using non-parametric comparisons Wilcoxon method. View Large Table 1. Effect of challenge with fresh (maintained at 4°C), aged (6 d at 45°C), or a ratio of aged/fresh Eimeria maxima oocysts at 10 days of age on d15 lesion scores and d10–d15 BWG as compared to non-challenged controls (Experiment 1). Eimeria maxima Challenge level %AF1 Post-challenge BWG2 (g) Gross lesion score3 Negative control N/A 225 ± 10.8 0.21 ± 0.11b 25,000 Aged 93.3 228 ± 6.9 0.30 ± 0.15b 25,000 Aged:fresh (60%:40%) 61.1 212 ± 10.6 1.20 ± 0.13a 25,000 Fresh 12.8 206 ± 5.33 0.86 ± 0.14a Eimeria maxima Challenge level %AF1 Post-challenge BWG2 (g) Gross lesion score3 Negative control N/A 225 ± 10.8 0.21 ± 0.11b 25,000 Aged 93.3 228 ± 6.9 0.30 ± 0.15b 25,000 Aged:fresh (60%:40%) 61.1 212 ± 10.6 1.20 ± 0.13a 25,000 Fresh 12.8 206 ± 5.33 0.86 ± 0.14a a,bMeans ± SEM within variable columns with different superscripts are significantly different (P < 0.05). 1AF = Autofluorescence. Determined using Zeiss Axio Imager M2 430004-9902 with filter set 38 1031-346 with an excitation of BP 470/40, beamsplitter of FT 495, and emission spectrum of BP 525/50. 2Although there were no significant differences, the negative control was compared to the 25,000 fresh using a one-way ANOVA (P = 0.054). 3Analyzed using non-parametric comparisons Wilcoxon method. View Large Experiment 2 Utilizing ANOVA, the fresh (7% AF) treatment group was detected as statistically different (P < 0.05) in post-challenge BWG when compared to the negative control and aged (99% AF) groups (Table 2). The ratio aged/fresh (80.6% AF) treatment group was determined to be statistically different (P < 0.05) from the negative control in post-challenge BWG. Both the ratio aged/fresh (80.6%AF) and fresh (7% AF) treatment groups were statistically different from the negative control and aged (99% AF) groups in LS. Table 2. Effect of challenge with fresh (maintained at 4°C), aged (6d at 45°C), or a ratio of aged:fresh Eimeria maxima oocysts at 10 days of age on d15 lesion scores, d10–d15 BWG, and mean total oocyst shedding from d15–18 per bird as compared to non-challenged controls (Experiment 2). Eimeria maxima Challenge level %AF1 Post-challenge BWG2 (g) Gross lesion score3 Mean total OPG/bird2 Negative control N/A 226 ± 4.95a 0.00 ± 0.00b 0 ± 0.00b 22,500 Aged 99 214 ± 7.72a,b 0.11 ± 0.11b 0 ± 0.00b 22,500 Aged:fresh (80%:20%) 80.6 197 ± 9.14b,c 1.42 ± 0.14a 24,569 ± 1,903a 22,500 Fresh 7 176 ± 6.25c 2.00 ± 0.10a 24,964 ± 2,334a Eimeria maxima Challenge level %AF1 Post-challenge BWG2 (g) Gross lesion score3 Mean total OPG/bird2 Negative control N/A 226 ± 4.95a 0.00 ± 0.00b 0 ± 0.00b 22,500 Aged 99 214 ± 7.72a,b 0.11 ± 0.11b 0 ± 0.00b 22,500 Aged:fresh (80%:20%) 80.6 197 ± 9.14b,c 1.42 ± 0.14a 24,569 ± 1,903a 22,500 Fresh 7 176 ± 6.25c 2.00 ± 0.10a 24,964 ± 2,334a a–cMeans ± SEM within variable columns with different superscripts are significantly different (P < 0.05). 1AF = Autofluorescence. Determined using Zeiss Axio Imager M2 430004-9902 with filter set 38 1031-346 with an excitation of BP 470/40, beamsplitter of FT 495, and emission spectrum of BP 525/50. 2Statistical evaluation using ANOVA followed by Tukey's multiple comparison post hoc test. 3Analyzed using non-parametric comparisons Wilcoxon method. View Large Table 2. Effect of challenge with fresh (maintained at 4°C), aged (6d at 45°C), or a ratio of aged:fresh Eimeria maxima oocysts at 10 days of age on d15 lesion scores, d10–d15 BWG, and mean total oocyst shedding from d15–18 per bird as compared to non-challenged controls (Experiment 2). Eimeria maxima Challenge level %AF1 Post-challenge BWG2 (g) Gross lesion score3 Mean total OPG/bird2 Negative control N/A 226 ± 4.95a 0.00 ± 0.00b 0 ± 0.00b 22,500 Aged 99 214 ± 7.72a,b 0.11 ± 0.11b 0 ± 0.00b 22,500 Aged:fresh (80%:20%) 80.6 197 ± 9.14b,c 1.42 ± 0.14a 24,569 ± 1,903a 22,500 Fresh 7 176 ± 6.25c 2.00 ± 0.10a 24,964 ± 2,334a Eimeria maxima Challenge level %AF1 Post-challenge BWG2 (g) Gross lesion score3 Mean total OPG/bird2 Negative control N/A 226 ± 4.95a 0.00 ± 0.00b 0 ± 0.00b 22,500 Aged 99 214 ± 7.72a,b 0.11 ± 0.11b 0 ± 0.00b 22,500 Aged:fresh (80%:20%) 80.6 197 ± 9.14b,c 1.42 ± 0.14a 24,569 ± 1,903a 22,500 Fresh 7 176 ± 6.25c 2.00 ± 0.10a 24,964 ± 2,334a a–cMeans ± SEM within variable columns with different superscripts are significantly different (P < 0.05). 1AF = Autofluorescence. Determined using Zeiss Axio Imager M2 430004-9902 with filter set 38 1031-346 with an excitation of BP 470/40, beamsplitter of FT 495, and emission spectrum of BP 525/50. 2Statistical evaluation using ANOVA followed by Tukey's multiple comparison post hoc test. 3Analyzed using non-parametric comparisons Wilcoxon method. View Large There was no detectible OS within the negative control and aged (99%AF) treatment groups, whereas significant numbers were detected within the ratio aged/fresh (80.6% AF) and fresh (7%AF) treatment groups (Table 2). The ratio aged/fresh (80.6% AF) and fresh (7% AF) treatment groups were found to have OS significantly different from the negative control and aged (99% AF) treatment groups. However, the lack of statistical difference between the ratio (80.6% AF) and fresh (7% AF) groups can potentially be explained by “crowding effect” as mentioned earlier (Johnston et al., 2001; Williams, 2001). Overall, it appeared that increased %AF was consistent with our hypothesis of non-viability within Eimeria oocysts. The treatment groups with higher %AF resulted in a general consistency of lower LS, higher BWG, and reduced OS as compared to the negative control and aged oocyst treatment groups—suggesting lowered disease challenge. This may suggest that AF could be a non-viability indicator, potentially offering an alternative method for easy enumeration for vaccine titration or research challenge studies. However, more research is needed among the various Eimeria species to determine whether AF is a consistent indicator of non-viability for all species. Moreover, although AF in fresh and aged oocysts has been associated with increased levels of dityrosine crosslinks (Belli et al., 2003, 2006), the AF substance(s) associated with accelerated aging in the present studies were not identified. REFERENCES Augustine P. C. 1980 . 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Poultry ScienceOxford University Press

Published: Jul 11, 2018

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