Evaluation of Eimeria adenoeides Preinfection on the Severity of Histomoniasis in Turkeys.

The objectives of this study were to evaluate whether a preinfection of Eimeria adenoeides (EAD) or Eimeria tenella (ET) could affect the severity of subsequent histomoniasis in turkeys (Experiment 1) and if previous exposure to EAD infection, when a single or multiple inoculations of EAD were administered with sufficient time for complete cecal recovery, would affect the severity of HM incidence and lesions (Experiment 2). In Experiment 1, 200 poults were assigned to 1 of 5 groups, as follows: unchallenged negative control, positive challenge control inoculated with 105 HM, EAD at 500 oocysts/bird and Histomonas meleagridis (HM), EAD at 2500 oocysts/bird and HM, or ET at 9 × 106 oocysts/bird and HM. ET and EAD were inoculated on day 15 and HM on day 20. In Experiment 2, the trial consisted of two different challenge ages to evaluate short- or long-term EAD effects before HM challenge. Poults (n = 260) were assigned to either early-HM-challenged groups (HM on day 19 challenge control or EAD at 2500 oocysts/bird on day 14 with HM on day 19) or late-HM-challenged groups (HM on day 35 challenge control, EAD at 2500 oocysts/bird on day 14 and HM on day 35, or EAD at 100 oocysts/bird every 2-3 days during the first 3 weeks and HM on day 35). An unchallenged negative-control group was used for both the early- and late-challenge phases in Experiment 2. Mortalities were recorded, and surviving poults were scored for histomoniasis-related hepatic and cecal lesions. In Experiment 1, preinfection with both doses of EAD reduced the mortality as well as the cecal and hepatic lesions caused by histomoniasis. In Experiment 2, neither short- nor long-term preinfection with EAD had an effect on histomoniasis-related mortality or lesions. Differences between Experiments 1 and 2 may be due to the level of infection caused by the prechallenge with EAD and the resulting destruction of cecal tissue.

Evaluación de la preinfección por Eimeria adenoeides sobre la severidad de la histomoniasis en pavos. Los objetivos de este estudio fueron evaluar si una preinfección por Eimeria adenoeides (EAD) o Eimeria tenella (ET) podría afectar la severidad de la histomoniasis subsequente en pavos (Experimento 1); y si la exposición previa a la infección por E. adenoeides, cuando se administraron una o varias inoculaciones de E. adenoeides con tiempo suficiente para la completa recuperación cecal, afectaría la gravedad de la incidencia y las lesiones de Histomonas meleagridis (Experimento 2). En el Experimento 1, se asignaron 200 pavipollos en cinco grupos, de la siguiente manera: control negativo no desafiado, control de desafío positivo inoculado con 105 de H. meleagridis, un grupo con E. adenoeides a 500 ooquistes/ave e H. meleagridis (HM), otro grupo con E. adenoeides a 2500 ooquistes/ave y H. meleagridis, o E. tenella a 9×106 ooquistes/ave y H. meleagridis. Se inocularon E. tenella y E. adenoeides el día 15 y H. meleagridis el día 20. En el Experimento 2, el ensayo consistió en dos edades de exposición diferentes para evaluar los efectos de E. adenoeides a corto o largo plazo antes del desafío con H. meleagridis. Los pavipollos (n = 260) se asignaron a los grupos de desafío temprano con H. meleagridis (H. meleagridis en el día 19 en el grupo control de desafío o E. adenoeides con 2500 ooquistes/ave el día 14 y con H. meleagridis en el día 19) o los grupos de desafío tardío con H. meleagridis (H. meleagridis en el día 35 del control de desafío, E. adenoeides a 2,500 ooquistes/ave el día 14 y H. meleagridis en el día 35, o E. adenoeides con 100 ooquistes/ave cada 2-3 días durante las primeras 3 semanas y H. meleagridis en el día 35). En el Experimento 2, se utilizó un grupo de control negativo no desafiado para ambas fases de exposición temprana y tardía. Se registraron la mortalidad y los pavipollos supervivientes se asignaron puntuaciones en cuanto a lesiones hepáticas y cecales relacionadas con histomoniasis. En el Experimento 1, la preinfección con ambas dosis de E. adenoeides redujo la mortalidad, así como las lesiones cecales y hepáticas causadas por histomoniasis. En el Experimento 2, ni la preinfección a corto ni a largo plazo con E. adenoeides tuvo un efecto sobre la mortalidad o las lesiones relacionadas con la histomoniasis. Las diferencias entre los Experimentos 1 y 2 pueden deberse al nivel de infección causado por el desafío previo con E. adenoeides y la destrucción resultante del tejido cecal.

Exploiting digital droplet PCR and Next Generation Sequencing technologies to determine the relative abundance of individual Eimeria species in a DNA sample.

DNA-based diagnostic assays for detecting infections with Eimeria species have been limited to providing identification and presence/absence data for samples containing oocysts. Modern technologies that generate quantitative data, such as droplet digital PCR (ddPCR) and Next Generation Sequencing (NGS), utilize a relatively short amplicon size containing sufficient species-specific variation for reliable species level identification. Targeting the cytochrome c oxidase subunit III gene in the mitochondrial genome, we established protocols using these technologies to determine the relative abundance of the number of copies/µL of Eimeria species in a sample. Samples from chickens of known and unknown Eimeria species composition were analyzed to determine the suitability of these technologies as diagnostic assays. All technologies demonstrated robust capability of identifying and quantifying the Eimeria species in samples. The new quantitative assays described herein will produce invaluable detail of Eimeria species infections for an array of situations in commercial chicken production systems, enabling further characterization of the disease profile and allowing for the development or enhancement of new intervention strategies.

Restoration of anticoccidial sensitivity to a commercial broiler chicken facility in Canada.

Increasing resistance of Eimeria species to anticoccidial medications is an issue in the broiler chicken industry. Using drug-sensitive strains in live-coccidiosis vaccines has been shown to improve anticoccidial effectiveness in US-based broiler production. In Canada, litter is removed between flocks, which differ from the US industry practice. Thus, we investigated the use of drug-sensitive vaccine strains in a Canadian broiler production facility with suspected anticoccidial resistance. Weekly fecal samples were collected from flocks before, during, and after vaccine seeding to determine oocyst shedding patterns; following the vaccine seeding, OPG counts from similar aged birds were lower than flocks before live-coccidiosis vaccine use. Eimeria species isolates, collected before and after vaccine seeding, were used in 2 anticoccidial sensitivity tests to evaluate their susceptibility to commercially available anticoccidial medications; a low-dose challenge to define parasite replication, and a high-dose challenge to monitor broiler performance. In both experiments, isolates collected after seeding were more susceptible to almost every anticoccidial medication evaluated compared with the isolates collected before seeding. These results demonstrate an improvement in sensitivity to many anticoccidials after the use of live-coccidiosis vaccines at this facility. However, the regulated removal of litter at the end of each flock required under Canadian broiler chicken production management rules could limit the establishment of vaccine-strain Eimeria species in broiler facilities and could shorten the longevity of improved drug sensitivity observed in this study.

Monitoring coccidia in commercial broiler chicken flocks in Ontario: comparing oocyst cycling patterns in flocks using anticoccidial medications or live vaccination.

Coccidiosis, the parasitic disease caused by Eimeria spp., is controlled during broiler chicken production through the inclusion of in-feed anticoccidial medications. Live-coccidiosis vaccination has become an increasingly common alternative to these medications. Monitoring infections with Eimeria spp. in flocks can be accomplished through determining the concentration of oocysts excreted in the fecal material (i.e., oocysts per gram; OPG). The purpose of our study was to sample commercial Ontario broiler chicken flocks at various times of the year to determine weekly OPG counts for flocks that use either an in-feed anticoccidial medication or a live-coccidiosis vaccine. Weekly sampling of 95 flocks from placement to market permitted documentation of oocyst cycling patterns typical of conventional and antibiotic-free flocks, and variation of these patterns in summer and winter. Medicated flocks had higher and later peak oocyst shedding compared with vaccinated flocks. Flocks reared in the summer peaked in oocyst shedding earlier than flocks reared in the winter. Despite what appears to be poorer coccidiosis control in the medicated flocks, the performance data were similar for these flocks compared with vaccinated flocks. This is the first study describing typical patterns of parasite shedding in Ontarian commercial broiler chicken flocks; these data will provide a baseline of expected Eimeria spp. infections in Canadian broiler chicken flocks to ensure optimal coccidiosis prevention.

Research Note: Evaluating fecal shedding of oocysts in relation to body weight gain and lesion scores during Eimeria infection.

Coccidiosis has been a pervasive disease within the poultry industry, with test parameters used to measure effectiveness of treatment strategies often being subjective or influenced by non-disease-related activity. Four experiments were completed, which examined several test parameters of coccidiosis, including body weight gain (BWG), lesion scores, and oocysts per gram of feces (OPG). Each experiment included at least 2 parameters for measuring coccidial infection in chickens and turkeys. In experiment 1, an inoculated control was measured against 3 anticoccidial groups, whereas in experiments 2 to 4, noninoculated and inoculated controls were compared via BWG and OPG. Lesion scores were also included in experiments 1, 3, and 4. Experiment 4 resulted in high correlation, via Pearson correlation coefficient, between BWG and OPG (r = -0.69), very high correlation between OPG and lesion score (r = 0.86), and moderate correlation between BWG and lesion score (r = -0.49). Lesion scores proved to be effective in confirming Eimeria infection, although they did not correlate well with BWG or OPG. Each parameter tended to provide more useful information when lined up with the Eimeria life cycle. Incorporation of OPG, with BWG and lesion scores, as test parameters to measure coccidiosis intervention strategies, provides a global description of disease that may not otherwise be observed with the 2 latter measurements alone.

Cystoisospora felis infection in a captive jaguar cub (Panthera onca) in Michoacán, México.

Cystoisospora felis (Wenyon 1923) was identified in a 3-month-old, captive jaguar (Panthera onca) presenting with signs of gastrointestinal distress. The cub was fed beef, chicken, and commercial diet. Examination of fresh feces detected large (47.8 µm × 35 µm) unsporulated oocysts. Sporulated oocysts contained 2 sporocysts, each with 4 sporozoites. Oocyst morphometrics agreed with published features of C. felis described from domestic felines. Partial mitochondrial cytochrome c oxidase subunit I (mtCOI) gene was PCR-amplified and sequenced; the resulting sequence showed 100% identity to a C. felis isolate from a domestic cat. This is the first molecularly confirmed report of C. felis infecting and producing clinically evident, enteric disease in a jaguar cub.

Full Mitochondrial Genome and Nuclear 18S rDNA Sequences Refine the Taxonomic Placement of Choleoeimeria taggarti n. comb. from the Prostate of Antechinus flavipes (Yellow-Footed Antechinus).

An unusual coccidian parasite was described previously from the prostate of a male Antechinus flavipes (family: Dasyuridae; common name: yellow-footed antechinus). Morphometrics and a partial nuclear 18S small subunit rDNA (18S rDNA) sequence were used to assign this parasite to the genus Eimeria; it was named Eimeria taggarti. We generated full nuclear 18S rDNA and mitochondrial genome sequences from this parasite and used the newly completed 18S rDNA and mitochondrial cytochrome c oxidase subunit I (COI) sequences to perform a more in-depth phylogenetic analysis. The parasite clustered closely with Choleoeimeria spp. and Acroeimeria spp. infecting herptiles in a well-supported clade that was the sister lineage to the Eimeriidae sensu stricto. The mitochondrial genome of this parasite contained 2 inverted segments compared to mitochondrial genomes from parasites in the Eimeriidae sensu stricto (i.e., Stieda body-possessing coccidia with 4 dizoic sporocysts); this mitochondrial genome arrangement was shared with the only Choleoeimeria species for which sequence data were available publicly. Examination of histological preparations and TEM images uncovered bivalvate sporocysts and otherwise confirmed previously described morphological features of the parasite. Based on our phylogenetic analyses and histological observations, we propose the generic reclassification of E. taggarti to Choleoeimeria taggarti n. comb.

Nucleotide-rich yeast extract fed to broiler chickens challenged with Eimeria: impact on growth performance, jejunal histomorphology, immune system, and apparent retention of dietary components and caloric efficiency1.

Nucleotide-rich yeast extract (YN) was investigated for its effects on growth performance, jejunal histomorphology and mucosal immunoglobulin A (IgA), immune organs weight and apparent retention (AR) of components in broiler chickens challenged with Eimeria. A total of 336 day-old male chicks (Ross x Ross 708) were placed in floor pens and provided a corn-soybean meal-based diet without or with YN (500 g/mt) (n = 14). On day 10, 7 replicates per diet were orally administered with 1 mL of sporulated E. acervulina and E. maxima oocysts and the rest (non-challenged control) administered equivalent distilled water creating a 2 × 2 factorial arrangement for the post-challenge period (day 11 to 35). Feed intake (FI), BWG, and FCR responses were measured in pre- and post-challenge periods. Excreta samples were collected on day 14 to 17 and 31 to 34 for oocyst count and AR of components, respectively. On day 15 and 35, 5 birds/pen were necropsied for intestinal samples. Spleen, bursa, and thymus weights were also recorded at both time points and breast yield on day 35. Diet had no effect (P > 0.05) on pre-challenge growth performance. Interaction (P = 0.046) between Eimeria and YN on FI was such that Eimeria challenge increased FI (day 11 to 35) in non-YN birds. There was no interaction (P > 0.05) between Eimeria and YN on other post-challenge responses. Eimeria reduced (P < 0.05) BWG, FCR, caloric efficiency, day 15 jejunal villi height and IgA concentration, and increased (P < 0.01) day 15 spleen weight. On day 35, YN increased bursa weight (1.57 vs. 1.78 mg/g BW, P = 0.04). There was a tendency for an interaction effect (P = 0.09) on day 35 thymus weight, such that in challenged birds, YN fed birds tended to have a lighter thymus relative to non-YN fed birds. In conclusions, independent of Eimeria challenge, supplemental YN had no effect on growth performance, caloric efficiency, and intestinal function but increased immune organ weights.

Cecal coccidiosis in turkeys: Comparative biology of Eimeria species in the lower intestinal tract of turkeys using genetically typed, single oocyst-derived lines.

Differentiating the Eimeria species causing cecal coccidiosis in turkeys is challenging. To obtain benchmark biological data for Eimeria gallopavonis Hawkins 1952 and Eimeria meleagridis Tyzzer 1929 and to support the stability of the species concept for each, genetically typed, single oocyst-derived lines of E. gallopavonis Weybridge strain and E. meleagridis USAR97-01 were used to redescribe the biological, pathological, and morphological features of these parasites. Oocysts of E. meleagridis and E. gallopavonis overlap in dimensions, but oocysts of the former have a single polar granule compared with multiple in the latter. Mature first-generation meronts of E. gallopavonis were observed histologically as early as 48 h post-inoculation alongside the villi in jejunum (before and after Meckel's diverticulum), ileum, cecal neck and rectum, but not cecal pouches. Three asexual cycles were observed suggesting that early workers apparently overlooked one asexual cycle. Examination of endogenous development of a culture labeled "Eimeria adenoeides Weybridge strain" suggested that this strain (found in a number of publications as a large oocyst strain of "Eimeria adenoeides") matched the species description of E. gallopavonis and so has been renamed herein. Macroscopic lesions induced by E. gallopavonis consisted of caseous material distally from posterior of the yolk stalk through the remaining intestinal tract, excluding the cecal pouches. For E. meleagridis, only the first asexual generation was observed outside of the cecal pouches within the jejunum around the yolk stalk. Second- and 3rd-generation asexual stages developed almost exclusively in the cecal pouches (but not cecal necks). Macroscopic lesions described for E. meleagridis were similar to those of E. adenoeides. Marked corrugation of the cecal serosal surface was observed. Cecal pouches contained creamy colored, caseous material varying from loose material to granular. Distinguishing features of the Eimeria species infecting the lower part of the small intestine are summarized in the present study, and new type specimens were designated for E. gallopavonis and E. meleagridis to provide a stable reference for future work with these parasites.

Responses of broiler chickens to Eimeria challenge when fed a nucleotide-rich yeast extract.

Nucleotide-rich yeast extract (YN) was investigated for effects on growth performance, jejunal physiology, and cecal microbial activity in Eimeria-challenged broiler chickens. A total of 360-day-old male chicks (Ross × Ross 708) were placed on floor pens and provided a corn-soybean meal-based diet without or with YN (500 g/MT; n = 12). On d 10, 6 replicates per diet were orally administered with 1 mL of E. acervulina and E. maxima sporulated oocysts and the rest (non-challenged control) were administered with 1 mL of distilled water. On d 15, 5 birds/pen were then necropsied for intestinal lesion scores, histomorphology and cecal digesta pH, short chain fatty acids (SCFA), and microbial community using Illumina Miseq platform. Supplemental YN improved (P = 0.01) Feed conversion ratio (FCR) during the prechallenge phase (d 0 to 10). In the postchallenge period (d 11 to 15), Eimeria depressed (P < 0.05) Body weight gain (BWG) relative to non-challenged birds, whereas YN-fed birds had a higher (P = 0.05) BWG compared to that of non-YN-fed birds. There was an interaction between YN and Eimeria on jejunal villi height (VH) (P = 0.001) and expression of cationic amino acid transporter 1(CAT1) (P = 0.04). Specifically, in the absence of Eimeria, YN-fed birds had a shorter VH (892 vs. 1,020 µm) relative to that of control but longer VH (533 vs. 447 µm) in the presence of Eimeria. With respect to CAT1, YN-fed birds had a higher (1.65 vs. 0.78) expression when subjected to Eimeria than when not challenged. Independently, Eimeria decreased (P < 0.01) the jejunal expression of maltase, Na glucose transporter 1 and occludin genes, ceca digesta abundance of genus Clostridium cluster XlVa and Oscillibacter but increased (P < 0.01) jejunal proliferating cell nuclear antigen and interleukin 10. Interaction between YN and Eimeria was observed for ceca digesta pH (P = 0.04) and total SCFA (P = 0.01) such that YN increased SCFA in the absence of Eimeria but reduced SCFA and increased pH in the presence of Eimeria. In summary, Eimeria impaired performance and gut function and shifted gut microbiome; YN improved performance independently, attenuated Eimeria damage on indices of gut function, and modulated cecal microbiome.

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

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.

Growth performance and gastrointestinal responses of broiler chickens fed corn-soybean meal diet without or with exogenous epidermal growth factor upon challenge with Eimeria.

Epidermal growth factor (EGF), a protein known for its mitogenic and anti-apoptotic effects was fed to broiler chickens to evaluate growth performance, gastrointestinal measurements, and apparent retention (AR) of components upon challenge with Eimeria. A total of 216, d old male broiler chicks (Ross 708) were placed in cages (6 birds/cage) and allocated to treatments. The treatments were: 1) control (Lactotobacilli lactis fermentation supernatant without EGF), 2) 80 µg of EGF/kg BW/d, and 3) 160 µg of EGF/kg BW/d. A basal antibiotic-free corn-soybean diet containing TiO2 was used. Birds were offered fresh feed with respective treatments on daily basis and had free access to drinking water for 14 d. On d 5, birds (6 replicates per treatment) were challenged with 1 mL of E. acervulina and E. maxima mixture via oral gavage and the other 6 replicates were given sham. Growth performance was measured in pre- (d 0 to 5) and post- (d 6 to 14) challenge periods. Two birds per cage were necropsied on d 10 for intestinal lesion scores and tissue samples for histomorphology and expression of select intestinal genes. Excreta samples for AR of components and oocyst shedding were taken d 10 to 13 and all birds were necropsied on d 14 for gastrointestinal weight. The EGF linearly (P < 0.05) increased BWG before challenge. There was no EGF and Eimeria interaction (P > 0.05) on growth performance, AR of GE, and intestinal histomorphology; the main effects were such that Eimeria depressed (P < 0.01) BWG, FCR, AR of DM, crude fat, and GE, and villi height to crypt depth ratio. An interaction between EGF and Eimeria (P < 0.05) on indices of gut function was such that EGF improved expression of genes for nutrient transporters and tight junction proteins in Eimeria challenged birds whilst no effect in non-challenged control. In conclusion, Eimeria challenge reduced growth performance and impaired gut function; EGF showed beneficial effects on growth pre-challenge and improved indices of gut function upon Eimeria challenge.

Re-description of a genetically typed, single oocyst line of the turkey coccidium, Eimeria dispersa Tyzzer, 1929.

The Briston strain of Eimeria dispersa Tyzzer, 1929 was isolated originally from a commercial turkey flock from Briston, Norfolk, UK. A single oocyst-derived line of E. dispersa was propagated and used to re-describe biological and morphological features of E. dispersa in the turkey. Oocysts of the Briston strain measured 26 ± 1.1 µm (24-28) by 21 ± 1 µm (19-23); these were larger than oocysts described originally by Tyzzer in 1929 (22.75 by 18.84 µm) but within dimensions (26.07 by 21.04 µm) reported by Hawkins (1952) in his description of E. dispersa isolated from turkeys. In the present study, endogenous development started mainly in duodenum and upper jejunum and then spread down toward the lower jejunum. A few parasites were detected in the ileum beginning 96 h post-infection; only few gamonts were observed in the cecal neck area at 120 h, and no parasites were detected in cecal pouches or rectum. Four asexual generations were observed before the start of gametogony, and only one large type of first generation meront was detected in duodenum and upper jejunum at 32 h. This strain has a prepatent period of 120 h. The Briston strain of E. dispersa is a mildly pathogenic coccidium. Duodenum and jejunum of infected birds were slightly dilated and paler in color than of uninfected controls. There was whitish green mucoid material in the lumen of the duodenum and jejunum. The mucosa looked slightly congested and edematous with a few scattered petechial hemorrhages.

The epizootiology of Eimeria infections in commercial broiler chickens where anticoccidial drug programs were employed in six successive flocks to control coccidiosis.

The course of natural Eimeria infections in 6 successive broiler flocks at a commercial farm comprising 4 houses, where different anticoccidial drug programs were employed, was studied by counting the number of oocysts in the litter at weekly intervals. The course of infection in all flocks followed a bell shaped curve in which oocyst numbers, initially low, increased to a peak ranging from 36 × 10(3) to 74 × 10(3) oocysts/g (OPG) of litter around 3 to 4 wk of age. Numbers subsequently declined to 3 × 10(3) to 15 × 10(3) OPG. Oocysts could be detected between flocks when birds were not present. Species of Eimeria identified included E. acervulina, E. maxima, and E. tenella Despite the presence of large numbers of oocysts in the litter, coccidial lesions were not observed in the intestines of the birds. The performance of broilers at the study site was comparable to that of other farms in the area where birds from the same settlement were reared to a similar age using the same drug programs. The results indicate the ubiquitous nature of Eimeria spp. infections in commercial broilers despite prophylactic medication.

Sequence-based genotyping clarifies conflicting historical morphometric and biological data for 5 Eimeria species infecting turkeys.

Unlike with Eimeria species infecting chickens, specific identification and nomenclature of Eimeria species infecting turkeys is complicated, and in the absence of molecular data, imprecise. In an attempt to reconcile contradictory data reported on oocyst morphometrics and biological descriptions of various Eimeria species infecting turkey, we established single oocyst derived lines of 5 important Eimeria species infecting turkeys, Eimeria meleagrimitis (USMN08-01 strain), Eimeria adenoeides (Guelph strain), Eimeria gallopavonis (Weybridge strain), Eimeria meleagridis (USAR97-01 strain), and Eimeria dispersa (Briston strain). Short portions (514 bp) of mitochondrial cytochrome c oxidase subunit I gene (mt COI) from each were amplified and sequenced. Comparison of these sequences showed sufficient species-specific sequence variation to recommend these short mt COI sequences as species-specific markers. Uniformity of oocyst features (dimensions and oocyst structure) of each pure line was observed. Additional morphological features of the oocysts of these species are described as useful for the microscopic differentiation of these Eimeria species. Combined molecular and morphometric data on these single species lines compared with the original species descriptions and more recent data have helped to clarify some confusing, and sometimes conflicting, features associated with these Eimeria spp. For example, these new data suggest that the KCH and KR strains of E. adenoeides reported previously represent 2 distinct species, E. adenoeides and E. meleagridis, respectively. Likewise, analysis of the Weybridge strain of E. adenoeides, which has long been used as a reference strain in various studies conducted on the pathogenicity of E. adenoeides, indicates that this coccidium is actually a strain of E. gallopavonis. We highly recommend mt COI sequence-based genotyping be incorporated into all studies using Eimeria spp. of turkeys to confirm species identifications and so that any resulting data can be associated correctly with a single named Eimeria species.

Re-description of a genetically typed, single oocyst line of the turkey coccidium, Eimeria adenoeides Moore and Brown, 1951.

The Guelph strain of Eimeria adenoeides was obtained from a commercial turkey flock in Ontario, Canada, in 1985. Single oocyst derived lines of E. adenoeides were propagated, and one of them used to re-describe biological and morphological features of E. adenoeides in the turkey. Oocysts of this strain are within the lower size ranges in the original species description reported by Moore and Brown (1951); oocysts of the Guelph strain averaged 18.7 ± 1.4 µm (16.7-22.5) by 14.3 ± 0.9 µm (13-16.2, n = 30) with a shape index (SI) of 1.3 ± 0.1. It is possible that the original species description was based, at least in part, on a mixed culture of two or more Eimeria species. Immature first-generation meronts of E. adenoeides Guelph strain were observed histologically at 32 h post-infection in the ileum and cecal neck. Early studies reported only two asexual generations suggested that first asexual cycle observed at 32 h post-infection was overlooked. In the present study, three asexual generations were observed before the start of gametogony. The Guelph strain is also characterized by a prepatent period of 112 h. The Guelph strain of E. adenoeides is a highly pathogenic coccidium that forms classic cecal lesions, including prominent caseous cecal cores, during moderate to severe infections. The maximum output of oocysts (1.77 × 10(7) per bird) was obtained from birds inoculated with 1 × 10(3) oocysts; maximum fecundity (1.55 × 10(5) oocyst shed per oocyst inoculated) was obtained with an inoculation of 1 × 10(2) oocysts, but fecundity dropped dramatically as the inoculation dose increased. To promote stability of the E. adenoeides species concept, neotype specimens (a parahapantotype slides series and phototype) have been designated and deposited for future reference.

Biological re-description of a genetically typed, single oocyst line of the turkey coccidium, Eimeria meleagrimitis Tyzzer 1929.

For the purpose of re-describing the Eimeria species that infect the turkey (Meleagris gallopavo) and to establish benchmark biological information linked to genetic markers for each species, a strain of Eimeria meleagrimitis Tyzzer 1929 was obtained from a litter sample from a turkey farm in Minnesota, USA in 2008. Multiple pure lines were derived by infecting turkey poults with a single oocyst; one of these lines was then used to re-describe biological and morphological features of E. meleagrimitis in the turkey and to designate a neotype of E. meleagrimitis in the turkey. Oocyst morphometrics of this line matched those of this species as originally described by Tyzzer (Am J Hyg 10:269-383, 1929). Three asexual generations of merogony (the first generation of meronts large in size and the second and third generations small) were detected in the intestines before the onset of gametogony; no developmental stages were detected in the cecal pouches. No mortality was induced by this line of E. meleagrimitis even when turkey poults were infected with high doses of oocysts (up to 5 × 10(5) oocysts/bird) and despite the ability of E. meleagrimitis to induce severe mucosal damage in the upper and middle duodenum. Macroscopic lesions were characterized to provide a graded lesion scoring guide that should assist assessment of the severity of infections with this species in infected turkeys. The pathogenicity of the strain was investigated, and a significant reduction in weight gain and feed conversion ratio was observed with doses of 10(4) oocysts/bird or more. The maximum yield of oocysts in the feces was obtained when birds were inoculated with 5 × 10(3) oocysts.

Sequencing the complete mitochondrial genome of Eimeria mitis strain USDA 50 (Apicomplexa: Eimeriidae) suggests conserved start positions for mtCOI- and mtCOIII-coding regions.

Four complete mitochondrial (mt) sequences from a single-oocyst-derived line of Eimeria mitis USDA 50 were obtained (three from cloned whole-genome PCR products, one from directly sequenced whole-genome PCR product). The mt genome is 6,408 bp long with three genes (CytB, cytochrome c oxidase subunit I (COI) and cytochrome c oxidase subunit III (COIII)) and many rDNA fragments (large subunit rDNA 13, small subunit rDNA 10); organisation was identical to other Eimeria sp. mt genomes. Conserved start codon positions for both COI and COIII are suggested for all Eimeria mt genomes; these start codon positions exist and may also be conserved, in related apicomplexan parasites. Within the three separate cloned PCR products of near-complete mt genomes, there were 26 nucleotide differences (collectively) compared to the directly sequenced mt genome. These changes appear to be base misincorporations during PCR. Direct sequencing of long PCR amplification products may be more likely to generate accurate mt genomic sequences than cloning and subsequent sequencing.

Coccidiosis in hihi/stitchbirds (Notiomystis cincta) due to coccidia of the Eimeriidae.

AIM: To describe the pathology of coccidiosis in hihi and to provide preliminary data on the taxonomy of the coccidia involved using molecular methods. METHODS: In an initial study from 1994 to 1997, gross and histopathological examinations were performed on 12 dead juvenile hihi from the National Wildlife Centre (NWC) at Mt. Bruce. In a second study during 2008-2010 DNA from sporulated oocysts and liver tissue was used for PCR analysis and sequencing. Faecal samples were also obtained from infected hihi from the NWC and examined for coccidial oocysts, which were then sporulated in the laboratory in 1994-1997 and 2007-2009. In addition, a post mortem was performed on a dead adult hihi from the NWC in 2008, and 18 archived hihi tissues from 11 individual birds stored at the Institute of Veterinary, Animal and Biomedical Sciences (IVABS) were used for DNA extraction. RESULTS: Severe gross and histopathological changes in the intestine and occasionally in the liver were found in the 12 dead birds examined. The morphological characteristics of the sporulated oocysts suggested that two types of coccidia were present. PCR analysis and sequencing of extracted DNA supported the existence of at least two different coccidia species in hihi. These were genetically more closely related to the genus Eimeria than to the morphologically similar genus Cystisospora (formerly Isospora) of mammals. In addition, one liver tissue sample that was examined post mortem was positive for at least two different coccidia species of the family Eimeriidae according to sequencing results, and the presence of extraintestinal coccidian stages was confirmed. CONCLUSIONS: Preliminary morphological and sequencing results suggest that two types of eimeriid coccidia are present and at least one of these commonly has extraintestinal stages. CLINICAL RELEVANCE: Coccidiosis in hihi is a serious disease capable of causing mortalities in juvenile and adult birds in captive situations. Treatment and control of the disease will be difficult as the extraintestinal stages of the organism are likely to be refractile to oral treatment.

The role of an early Salmonella Typhimurium infection as a predisposing factor for necrotic enteritis in a laboratory challenge model.

Necrotic enteritis (NE) caused by Clostridium perfringens (CP) in poultry is an important bacterial disease in terms of economic implications. The disease is multifactorial and is invariably associated with predisposing factors. In the present experiments, we investigated the potential predisposing role of neonatal Salmonella Typhimurium (ST) infection for NE-associated mortality in a laboratory challenge model. In two experiments, day-of-hatch chicks were randomly assigned to four groups: Group 1, nonchallenged control; Group 2, chickens received Eimeria maxima (EM) and CP; Group 3, chickens received EM and CP and were also challenged with ST at day 1 of age; Group 4, chickens received EM and CP and were also challenged with ST at day 17 of age. Challenged groups received an oral dose of EM at 18 days of age and CP (10(8) colony-forming units/chick) at 22-23 days of age. When compared to EM and CP, chicks challenged with ST (day 1) had increased NE-associated mortality and CP-associated lesion scores (P < 0.05) in both experiments. Furthermore, body weight and body weight gain were lower (P < 0.05) in chicks infected with ST (day 1) in the first experiment, even though no differences (P > 0.05) were observed in weight gain in the second experiment. Chicks challenged with ST (day 17) were similar to the EM and CP group in all of the above-mentioned parameters, indicating that a paratyphoid infection in younger chicks remarkably alters the susceptibility to secondary bacterial infections. Based on this work, the authors suggest that an ST infection early in the age of a chick may be important for altering susceptibility to NE, an observation that may be useful from the perspective of experimental reproduction of this disease and, perhaps, as an economically important reason to address the problem of paratyphoid Salmonella infections in young chicks.