Developing an experimental necrotic enteritis model in turkeys - the impact of Clostridium perfringens, Eimeria meleagrimitis and host age on frequency of severe intestinal lesions.

BACKGROUND: Necrotic enteritis is a significant problem to the poultry industry globally and, in Norway up to 30% of Norwegian turkey grow-outs can be affected. However, despite an awareness that differences exist between necrotic enteritis in chickens and turkeys, little information exists concerning the pathogenesis, immunity, microbiota or experimental reproduction of necrotic enteritis in turkeys. In particular, it is important to determine the appearance of the gross lesions, the age dependency of the disease and the role of netB toxin of Clostridium perfringens. To this end, we report our findings in developing an in vivo experimental model of necrotic enteritis in turkeys. RESULTS: A four tier (0-3) scoring system with clearly defined degrees of severity of macroscopic intestinal lesions was developed, based on 2312 photographic images of opened intestines from 810 B.U.T. 10 or B.U.T. Premium turkeys examined in nine experiments. Loss of macroscopically recognizable villi in the anterior small intestine was established as the defining lesion qualifying for a score 3 (severe intestinal lesions). The developed scoring system was used to identify important factors in promoting high frequencies of turkeys with severe lesions: a combined Eimeria meleagrimitis and Clostridium perfringens challenge, challenge at five rather than 3 weeks of age, the use of an Eimeria meleagrimitis dose level of at least 5000 oocysts per bird and finally, examination of the intestines of 5-week-old turkeys at 125 to 145 h after Eimeria meleagrimitis inoculation. Numbers of oocysts excreted were not influenced by Clostridium perfringens inoculation or turkey age. Among three different lesion score outcomes tested, frequency of severe lesions proved superior in discriminating between impact of four combinations of Clostridium perfringens inoculation and turkey age at challenge. CONCLUSIONS: This study provides details for the successful establishment of an in vivo model of necrotic enteritis in turkeys.

Multilocus sequencing of Hepatozoon cf. griseisciuri infections in Ontario eastern gray squirrels (Sciurus carolinensis) uncovers two genotypically distinct sympatric parasite species.

Intra-leukocytic gamonts consistent with the description of Hepatozoon griseisciuri Clark, 1958 are reported for the first time in Canadian eastern gray squirrels (Sciurus carolinensis Gmelin, 1788). Polymerase chain reaction (PCR) amplification and direct Sanger sequencing identified a pair of distinct genotypes at both a nuclear and mitochondrial locus; two 18S ribosomal RNA gene sequences (rDNA; genotype A and genotype B: 1816 base pairs (bp); 98.8% pairwise identity) and 2 distinct complete mitochondrial genome sequences (genotype A: 6311 bp; genotype B: 6114 bp; 89.1% pairwise identity) were obtained from 3 H. griseisciuri-infected squirrels sampled in Guelph, Ontario. The genetic content of both circular-mapping mitochondrial genomes was conventional for apicomplexan protists; each encoded for 3 protein-coding genes (cytochrome c oxidase subunit I (COI); cytochrome c oxidase subunit III (COIII); and cytochrome B (CytB)), 14 fragmented large subunit rDNA, 10 fragmented small subunit rDNA, and 8 unassigned rDNA. These genotypes, based on sequences obtained from a pair of loci from two parasite genomes, confirm the presence of at least two Hepatozoon species infecting Ontario eastern gray squirrels, one of which is likely to be conspecific with H. griseisciuri.

Mitochondrial Junction Region as Genotyping Marker for Cyclospora cayetanensis.

Cyclosporiasis is an infection caused by Cyclospora cayetanensis, which is acquired by consumption of contaminated fresh food or water. In the United States, cases of cyclosporiasis are often associated with foodborne outbreaks linked to imported fresh produce or travel to disease-endemic countries. Epidemiologic investigation has been the primary method for linking outbreak cases. A molecular typing marker that can identify genetically related samples would be helpful in tracking outbreaks. We evaluated the mitochondrial junction region as a potential genotyping marker. We tested stool samples from 134 laboratory-confirmed cases in the United States by using PCR and Sanger sequencing. All but 2 samples were successfully typed and divided into 14 sequence types. Typing results were identical among samples within each epidemiologically defined case cluster for 7 of 10 clusters. These findings suggest that this marker can distinguish between distinct case clusters and might be helpful during cyclosporiasis outbreak investigations.

Distribution and abundance of Eimeria species in commercial turkey flocks across Canada.

Diversity and regional abundance of Eimeria species infecting Canadian commercial turkey flocks are largely unknown. To address this paucity of data regarding coccidiosis and its distribution in Canada, fecal samples from turkey flocks (N = 39) representing 27 commercial farms [ON (n = 20), SK (n = 2), BC (n = 3), AB (n = 1), NS (n = 1)] were screened for coccidia. Identification of all Eimeria species present in each sample was accomplished using a nested-polymerase chain reaction (PCR) assay targeting the mitochondrial cytochrome C oxidase subunit I gene. Most samples (33/39) were Eimeria-positive with 6 Eimeria species identified by the nested-PCR assay (1 to 6 species/sample, average 3.2); 4 samples (4/39, > 10% of samples) contained all 6 species. Eimeria species were common and distributed widely in Canadian commercial turkey flocks. Turkeys reared using in-feed medication or live vaccination for coccidiosis control had similar Eimeria species diversity within individual flocks. These preliminary observations highlight that coccidiosis remains a concern for Canadian turkey producers.

Distribution et abondance d'espèces d' Eimeria infectant des troupeaux de dindes commerciaux à travers le Canada. La diversité et l'abondance régionale d'espèces d'Eimeria infectants des troupeaux de dindes commerciaux canadiens sont pour la plupart inconnues. Pour adresser cette pénurie de données concernant les coccidies et leurs distributions au Canada, des échantillons fécaux provenant de 39 troupeaux de dindes, représentants 27 fermes commerciales [ON (n = 20), SK (n = 2), BC (n = 3), AB (n = 1), NS (n = 1)] étaient cribler pour la coccidie. L'identification de toutes les espèces d'Eimeria trouvées dans chaque échantillon était accomplie en utilisant une PCR nichée pour cibler la sous-unité I mitochondriale du cytochrome C oxydase. La plupart des échantillons (33/39) était positif pour l'Eimeria avec six espèces d'Eimeria identifiées par la PCR nichée (1 à 6 espèces/échantillon, moyenne 3,2); quatre échantillons (4/39, > 10 % d'échantillons) contenaient toutes les six espèces. Les espèces d'Eimeria sont communes et sont largement distribuées dans les troupeaux de dindes commerciaux canadiens. Les dindes élevées en utilisant des anticoccidiens en additifs alimentaire ou vaccinées avec des vaccins vivants pour la coccidie avaient une diversité d'espèces d'Eimeria similaire entre les troupeaux individuels. Ces observations préliminaires indiquent que la coccidie demeure toujours une préocculation pour les éleveurs de dindons.(Traduit par Alex Léveillé et Lisa Gordon).

Babesia odocoilei as a cause of mortality in captive cervids in Canada.

Nine cases of fatal infection with Babesia odocoilei were confirmed in reindeer (Rangifer tarandus tarandus) and elk (Cervus canadensis) housed in zoological institutions located in southern Quebec, Ontario, and Manitoba, Canada between 2013 and 2016. All animals died of a hemolytic crisis. Frequent postmortem findings were extensive hemorrhage, pigmenturia, and intrahepatic cholestasis. The described ante- and postmortem signs are consistent with those of previously reported cases in the United States. Diagnosis was confirmed in all cases by polymerase chain reaction performed on DNA extracted from whole blood or frozen spleen. We propose that babesiosis is an emerging disease of cervids in multiple Canadian provinces, most likely as a result of climate change and the northward range expansion of Ixodes scapularis, the primary tick vector for B. odocoilei. The role of captive animals as sentinels for wildlife health is also highlighted.

Babesia odocoilei,une cause de la mortalité chez les cervidés captifs au Canada. Entre 2013 à 2016, neuf cas d'infection fatale par Babesia odocoilei ont été détectés chez des caribous (Rangifer tarandus tarandus) et des wapitis (Cervus canadensis) gardés dans des établissements zoologiques situés dans le sud du Québec, de l'Ontario et du Manitoba, Canada. Les animaux sont morts suite à une crise hémolytique. Hémorragies, pigmenturie et cholestase intrahépatique ont fréquemment été identifiées à l'examen postmortem. Les signes ante- et postmortem décrits correspondent avec ceux des cas précédemment signalés aux États-Unis. Le diagnostic de babésiose fut confirmé par réaction en chaîne par polymérase sur l'ADN extrait d'échantillons de sang ou de rate congelée. Nous proposons que la babésiose des cervidés est une maladie émergente au Canada, et ce probablement en conséquence du réchauffement climatique et du mouvement vers le nord de la tique Ixodes scapularis, le principal vecteur de B. odocoilei. La valeur des animaux captifs comme sentinelles pour la santé de la faune est également discutée.(Traduit par les auteurs).

Live Eimeria vaccination success in the face of artificial non-uniform vaccine administration in conventionally reared pullets.

Live Eimeria vaccines against coccidiosis in poultry initiate immunity using a vaccine dose containing few oocysts; protection is enhanced through subsequent faecal-oral transmission ("cycling") of parasites in the poultry house. Spray-administered Eimeria vaccines can permit wide variations in doses ingested by individual chicks; some chicks may receive no primary vaccination at all. Consequently, protective immunity for the entire flock depends on successful environmental cycling of vaccine progeny. Pullets missing primary vaccination at day of age can become protected from coccidial challenge through cycling of vaccine progeny oocysts from vaccinated (V) cage mates. This study tested whether 40% cage floor coverage (CFC) with a durable material could improve protection against challenge in these "contact-vaccinated" (CV) or successfully V pullets. The six treatment groups tested were CV, V or sham-vaccinated pullets cage-reared on either 0% or 40% CFC. Oocyst output was measured separately for each group for 30 days following vaccine administration. Lesion scores, body weights and total oocyst outputs were measured to quantify protection at 30 days of age against single or mixed Eimeria species challenge infections. Use of 40% CFC to promote low-level oocyst cycling impacted the flock in two ways: (1) more uniform flock immunity was achieved in the 40% CFC (CV similar to V pullets) compared with 0% CFC and (2) protection was enhanced in the 40% CFC compared with the 0% CFC. The use of CFC is an easily adopted means of improving live Eimeria vaccination of caged pullets.

Molecular phylogenetics of eimeriid coccidia (Eimeriidae, Eimeriorina, Apicomplexa, Alveolata): A preliminary multi-gene and multi-genome approach.

Coccidia possess three distinct genomes: nuclear, mitochondrial, and plastid. Sequences from five genes located on these three genomes were used to reconstruct the phylogenetic relationships of members of the phylum Apicomplexa: 18S rDNA sequences from the nuclear (nu) genome, partial cytochrome c oxidase subunit I sequences from the mitochondrial (mt) genome, and partial 16S and 23S rDNA sequences and RNA polymerase B sequences from plastid (pl) genomes. Maximum parsimony, maximum likelihood, and Bayesian inference were used in conjunction with nuclear substitution models generated from data subsets in the analyses. Major groups within the Apicomplexa were well supported with the mitochondrial, nuclear, and a combination of mitochondrial, nuclear and concatenated plastid gene sequences. However, the genus Eimeria was paraphyletic in phylogenetic trees based on the nuclear gene. Analyses using the individual genes (18S rDNA and cytochrome c oxidase subunit I) resolved the various apicomplexan groups with high Bayesian posterior probabilities. The multi-gene, multi-genome analyses based on concatenated nu 18S rDNA, pl 16S, pl 23S, pl rPoB, pl rPoB1, and mt COI sequences appeared useful in resolving phylogenetic relationships within the phylum Apicomplexa. Genus-level relationships, or higher, appear best supported by 18S rDNA analyses, and species-level analyses are best investigated using mt COI sequences; for parasites for which both loci are available, nuclear 18S rDNA sequences combined with mitochondrial COI sequences provide a compact and informative molecular dataset for inferring the evolutionary relationships taxa in the Apicomplexa.

Simultaneous identification and DNA barcoding of six Eimeria species infecting turkeys using PCR primers targeting the mitochondrial cytochrome c oxidase subunit I (mtCOI) locus.

Species-specific PCR primers targeting the mitochondrial cytochrome c oxidase subunit I (mtCOI) locus were generated that allow for the specific identification of the most common Eimeria species infecting turkeys (i.e., Eimeria adenoeides, Eimeria meleagrimitis, Eimeria gallopavonis, Eimeria meleagridis, Eimeria dispersa, and Eimeria innocua). PCR reaction chemistries were optimized with respect to divalent cation (MgCl2) and dNTP concentrations, as well as PCR cycling conditions (particularly anneal temperature for primers). Genomic DNA samples from single oocyst-derived lines of six Eimeria species were tested to establish specificity and sensitivity of these newly designed primer pairs. A mixed 60-ng total DNA sample containing 10 ng of each of the six Eimeria species was used as DNA template to demonstrate specific amplification of the correct product using each of the species-specific primer pairs. Ten nanograms of each of the five non-target Eimeria species was pooled to provide a non-target, control DNA sample suitable to test the specificity of each primer pair. The amplifications of the COI region with species-specific primer pairs from pooled samples yielded products of expected sizes (209 to 1,012 bp) and no amplification of non-target Eimeria sp. DNA was detected using the non-target, control DNA samples. These primer pairs specific for Eimeria spp. of turkeys did not amplify any of the seven Eimeria species infecting chickens. The newly developed PCR primers can be used as a diagnostic tool capable of specifically identifying six turkey Eimeria species; additionally, sequencing of the PCR amplification products yields sequence-based genotyping data suitable for identification and molecular phylogenetics.

Description of two new Isospora species causing visceral coccidiosis in captive superb glossy starlings, Lamprotornis superbus (Aves: Sturnidae).

Isospora greineri sp. n. and Isospora superbusi sp. n. are described from captive superb glossy starlings, Lamprotornis superbus, from the Toronto Zoo succumbing to visceral coccidiosis. Sequence data from nuclear 18S recombinant DNA (rDNA) and mitochondrial cytochrome c oxidase subunit I (COI) loci from sporulated oocysts and infected tissues (liver, lung, or spleen) demonstrated two distinct Isospora sp. genotypes that varied in their relative abundance. In the tissues of one affected bird, as well as its associated fecal sample, two distinct COI sequences (1.7% divergence) and two distinct 18S rDNA sequences (0.6% divergence) were found at almost the same abundance; in other specimens, one of the 18S and one of the COI sequences were less abundant than the other. In the tissues of some birds, only a single COI and single 18S sequence were present. In all cases, the same pair of 18S rDNA and COI sequences fluctuated in abundance in parallel, indicating that there were two distinct species present rather than one species with more than one COI or 18S locus. The oocysts of these new species cannot be differentiated morphologically. Sporulated oocysts of both were spherical to subspherical measuring 17.7 ± 0.22 µm by 17.1 ± 0.20 µm with a mean L/W ratio of 1.03 ± 0.004. Sporocysts were ovoid measuring 13.5 ± 0.17 µm by 9.3 ± 0.15 µm with a mean L/W ratio of 1.4 ± 0.02. Sporocysts had a small Stieda body with indistinct sub-Stieda body; each sporocyst had a compact residuum. Two morphologically similar but genetically divergent Isospora species were shown to cause simultaneous enteric and extraintestinal infections in captive superb glossy starlings.

CORRECTING THE NAMES OF TWO HAEMOGREGARINA SPP. FROM LIZARDS IN EGYPT.

Two haemogregarine "species" names, Haemogregarina tarentannulari and Haemogregarina rawashi, were cited by Saoud et al. (1995) as having been described by Mohammed and Ramadan (1996, in press). However, the paper by Mohammed and Ramadan (1996) was never published and, therefore, these names and their authorities must be suppressed because they violate Chapter 3 (Criteria for Publication), Article 8 (What Constitutes Published Work) of the International Code of Zoological Nomenclature. The following new names are introduced to replace them based on the Principle of Priority (Chapter 6, Article 23, 23.1, and Chapter 11, Article 51, Recommendation 51E): Hepatozoon rawashi (Mohammed and Ramadan in Saoud, Ramadan, Mohammed and Fawzi, 1995) n. comb., with gamonts in the erythrocytes and meronts in the lungs and liver of the fan-footed gecko, Ptyodactylus hasselquisiti (Donndorff, 1798) from Egypt, and Haemogregarina tarentannulari (Mohammed and Ramadan in Saoud, Ramadan, Mohammed and Fawzi, 1995), with gamonts in the erythrocytes and meronts in the lungs and liver of the white-spotted wall gecko, Tarentola annularis (St. Hilaire, 1827) also from Egypt. This latter species was determined to be a junior synonym of Haemogregarina annularis El-Naffar, Mandour, and Mohammed 1991, which was later reassigned to the genus Hepatozoon based on their phylogenetic analysis of 18S rDNA gene sequences.

Growth performance, organs weight, intestinal histomorphology, and oocyst shedding in broiler chickens offered novel single strain Bacillus subtilis isolated from camel dung and challenged with Eimeria.

We evaluated a single strain Bacillus subtilis BS-9 direct-fed microbial (BSDFM) isolated from camel dung in Eimeria challenged broiler chickens. Seven-hundred d-old Ross 708 male chicks were placed in pens (25 birds/pen) and allocated to 2 treatments (n = 14). From d 0 to 13, control pens received untreated water (-BSDFM), and 2 treated pens received water and 2 mL x 108 colony forming unit/bird/d (+BSDFM); daily water intake (WI) was recorded. On d 9, birds in half (+Eimeria) of pens per treatment received of 1 mL of Eimeria maxima and Eimeria acervulina oocysts orally, and the other half (-Eimeria) sterile saline solution. Birds had ad libitum access to feed and a water line from d 14. Feed intake (FI), body weight (BW) and mortality were recorded for calculating BW gain (BWG) and feed conversion ratio (FCR). On d 14 and 35, samples of birds were necropsied for organ weight and intestinal measurements. Excreta samples were collected from d 14 to 19 for oocyst count. There was no treatment effect (P > 0.05) on growth performance or WI on d 0 to 9. There were interactions between BSDFM and Eimeria on d 19 (P = 0.014) and 29 (P = 0.036) BW with unchallenged +BSDFM birds being heavier than birds in the other treatments. The main effects (P < 0.05) on d 10 to 35 FI, BW, and BWG were such that +BSDFM increased and Eimeria decreased (P < 0.01) these parameters. There was interaction (P = 0.022) between BSDFM and Eimeria on d 10 to 35 FCR such that the FCR of challenged -BSDFM birds was poor than that of unchallenged counterparts, but none differed with +BSDFM birds. There was an interaction (P = 0.039) between BSDFM and Eimeria on d 14 bursa weight with challenged birds exhibiting heavier bursa than unchallenged +BSDFM birds. Eimeria reduced (P = 0.01) and BSDFM (P = 0.002) increased the villi height to crypt depth ratio. Results showed that BSDFM supplementation via water can support the growth performance of broiler chickens challenged with Eimeria and may be a strategy to reduce adverse effects of coccidiosis.

Using microsphere or fluorescein tracers and total oocyst output to measure ingestion of material following live-coccidiosis vaccinations.

One method of prevention of coccidiosis in broiler chickens raised without antibiotics relies on coccidiosis vaccination. Live-coccidiosis vaccines carry the risk for pathogenic effects if the Eimeria species overcycle. However, all chicks must receive an appropriate dose of Eimeria oocysts to induce immunity and reduce the risk of adverse effects. At the hatchery, coccidiosis vaccines are administered topically to boxes of chicks by spray or gel-droplet application. Determining the volume of vaccine ingested by individual chicks could provide a means of evaluating the success of different application methods. For each of 2 mass application methods (spray, gel-droplet), we used 3 quantification methodologies to determine the amount of vaccine material ingested by chicks: total oocyst counts from feces collected 5- to 8-days postvaccination; and counts of either microsphere or fluorescein tracers recovered from the gastrointestinal tract 30-min postvaccination. For each quantification methodology, chicks vaccinated via spray or gel-droplet application were compared to chicks vaccinated via oral gavage using the same concentration of oocysts per mL for all groups. Chicks vaccinated via gel-droplet application shed 10-fold more oocysts than those vaccinated by spray application. Individual chick consumption of vaccine material using tracers also revealed that chicks ingested more material following gel-droplet application than spray application, although the magnitude of the difference varied based on quantification methodology. The results of this study suggest that all 3 quantification methodologies can be used to help validate and improve mass vaccine application methods to ensure optimal ingestion, and therefore, coccidiosis vaccination success.

A NEW EIMERIA SPECIES (APICOMPLEXA: EIMERIIDAE) PARASITIZING COMMERCIAL CHUKAR PARTRIDGES (ALECTORIS CHUKAR) IN SOUTHERN ONTARIO.

A commercial producer hatching and rearing chukar partridges (Alectoris chukar) in Ontario, Canada had flocks experiencing coccidiosis. Microscopic analysis of Eimeria species isolated from a field sample indicated the presence of 2 distinct oocyst morphotypes; the most abundant species was determined to be Eimeria chapmani, based on oocyst morphology and sequence-based genotyping, and the less abundant, second Eimeria sp. was an undescribed parasite. Oocysts of the unknown Eimeria sp. were large and oval-shaped; dimensions averaged 27.9 µm by 17.0 µm (shape index = 1.65 µm). Oocysts contained at least 1 polar granule and 4 almond-shaped sporocysts with average dimensions measuring 12.5 µm by 6.9 µm (shape index = 1.83). Each sporocyst featured a Stieda body, sub-Stieda body, and sporocyst residuum; a sporocyst contained 2 sporozoites that each possessed a small anterior refractile body and a larger posterior refractile body. Virtually all oocysts sporulated after 24 hr when suspended in potassium dichromate at room temperature (22 C) on a rotary platform. Experimental infections with various doses of oocysts demonstrated elevated parasite shedding from birds gavaged with higher challenge doses; fecundity generally decreased in heavier infections. The approximate prepatent period of the parasite was 4-5 days (unsporulated oocysts observed histologically at 90 hr postinfection and in feces by day 5) and patency lasted until day 12 postinfection. To characterize the endogenous development of the Eimeria sp., tissues were collected at 8 regions along the intestinal tract (including the ceca and rectum) every 6 hr throughout the estimated prepatent period. Parasites were observed to infect the descending and ascending duodenum, midjejunum, proximal and distal ileum, and the ceca. The endogenous stages identified included intracellular sporozoites, 3 generations of merogony, and gametogonic stages. Sequences of the mitochondrial genome (GenBank MW934555) and nuclear 18S ribosomal DNA (GenBank MW934259) were obtained using polymerase chain reaction amplification for Sanger sequencing, and these were unique from all published sequences on GenBank. Molecular data, in conjunction with the unique biology of the Eimeria sp. isolated from the chukar partridge flock, support that this coccidium is new to science.

In vitro and in vivo evaluation of chlorhexidine salts as potential alternatives to potassium dichromate for Eimeria maxima M6 oocyst preservation.

Introduction: Coccidiosis caused by the Eimeria spp., an Apicomplexan protozoon, is a major intestinal disease that affects the poultry industry. Although most cases of coccidiosis are subclinical, Eimeria infections impair bird health and decrease overall performance, which can result in compromised welfare and major economic losses. Viable sporulated Eimeria oocysts are required for challenge studies and live coccidiosis vaccines. Potassium dichromate (PDC) is typically used as a preservative for these stocks during storage. Although effective and inexpensive, PDC is also toxic and carcinogenic. Chlorhexidine (CHX) salts may be a possible alternative, as this is a widely used disinfectant with less toxicity and no known carcinogenic associations. Methods: In vitro testing of CHX gluconate and CHX digluconate exhibited comparable oocyst integrity and viability maintenance with equivalent bacteriostatic and bactericidal activity to PDC. Subsequent use of CHX gluconate or digluconate-preserved Eimeria oocysts, cold-stored at 4°C for 5 months, as the inoculum also resulted in similar oocyst shedding and recovery rates when compared to PDC-preserved oocysts. Results and discussion: These data show that using 0.20% CHX gluconate could be a suitable replacement for PDC. Additionally, autofluorescence was used as a method to evaluate oocyst viability. Administration of artificially aged oocysts exhibiting >99% autofluorescence from each preserved treatment resulted in no oocyst output for CHX salt groups.

Phylogenetic position of the adeleorinid coccidia (Myzozoa, Apicomplexa, Coccidia, Eucoccidiorida, Adeleorina) inferred using 18S rDNA sequences.

Investigating the evolutionary relationships of the major groups of Apicomplexa remains an important area of study. Morphological features and host-parasite relationships continue to be important in the systematics of the adeleorinid coccidia (suborder Adeleorina), but the systematics of these parasites have not been well-supported or have been constrained by data that were lacking or difficult to interpret. Previous phylogenetic studies of the Adeleorina have been based on morphological and developmental characters of several well-described species or based on nuclear 18S ribosomal DNA (rDNA) sequences from taxa of limited taxonomic diversity. Twelve new 18S rDNA sequences from adeleorinid coccidia were combined with published sequences to study the molecular phylogeny of taxa within the Adeleorina and to investigate the evolutionary relationships of adeleorinid parasites within the Apicomplexa. Three phylogenetic methods supported strongly that the suborder Adeleorina formed a monophyletic clade within the Apicomplexa. Most widely recognized families within the Adeleorina were hypothesized to be monophyletic in all analyses, although the single Hemolivia species included in the analyses was the sister taxon to a Hepatozoon sp. within a larger clade that contained all other Hepatozoon spp. making the family Hepatozoidae paraphyletic. There was an apparent relationship between the various clades generated by the analyses and the definitive (invertebrate) host parasitized and, to lesser extent, the type of intermediate (vertebrate) host exploited by the adeleorinid parasites. We conclude that additional taxon sampling and use of other genetic markers apart from 18S rDNA will be required to better resolve relationships among these parasites.

The First Isospora Species (Apicomplexa: Eimeriidae) Described from the Northern Yellow-Shafted Flicker (Colaptes auratus luteus) in Ontario, Canada.

PURPOSE: Members of the genus Isospora commonly infect a wide variety of wild birds. By combining morphometric and genetic data, we are able to differentiate and describe a new apicomplexan parasite, Isospora picoflavae n. sp., from the Northern Yellow-shafted Flicker (Colaptes auratus luteus) in Ontario, Canada. METHODS: Unsporulated oocysts were observed in intestinal contents obtained during necropsy; these oocysts completed exogenous sporulation within 7 days at room temperature. Complete nuclear 18S rDNA and complete mitochondrial genome sequences were obtained from this previously undescribed Isospora sp. to compare with related coccidia. RESULTS: Sporulated oocysts were subspherical in shape measuring an average of 22.7 × 21.7 µm (n = 53; mean shape index 1.05) containing a near-spherical polar granule. Sporocysts were ovoidal to ellipsoidal in shape, measuring an average size of 17.0 × 10.5 µm (n = 9; mean shape index 1.62). Sporocyst residuum was concentrated in an irregular, spherical mass in the middle of each sporocyst. The isolated oocysts differed morphologically from other Isospora species reported to infect members of the family Picidae. Based on phylogenetic analyses using either combined nuclear 18S rDNA and mitochondrial cytochrome c oxidase I (COI) sequences or complete mitochondrial genome sequences, this Isospora sp. n. isolated from the Northern Flicker grouped within a clade containing Isospora species described from various passeriform hosts. CONCLUSION: Based on combined morphological and molecular data, the oocysts found in the gastrointestinal contents of Colaptes auratus luteus represent a new species of Isospora named herein as Isospora picoflavae n. sp.

MORPHOLOGICAL AND MOLECULAR CHARACTERIZATION OF ENTERIC COCCIDIA ISOLATED FROM BLACK-FOOTED FERRETS (MUSTELA NIGRIPES).

Black-footed ferrets (BFF; Mustela nigripes) are the only ferret species native to North America and have been listed as endangered since 1967. Starting in 1986, a multi-institutional effort has been breeding this species in captivity with successful reintroductions back into the wild. Enteric coccidiosis is recognized as a frequent cause of juvenile morbidity and mortality in captive breeding programs, and can result in substantial population losses. Despite this, little is known about the etiology of coccidiosis in BFF. Coccidia-positive fecal samples (n=12) and formalin-fixed paraffin-embedded intestinal tissues (n=11) were obtained from BFF in the Toronto Zoo (Ontario, Canada) and Louisville Zoo (Kentucky, USA) Species Survival Plan (SSP) populations. Oocyst morphometrics and sequence genotyping at three loci (nuclear 18S rDNA, mitochondrial cytochrome c oxidase subunit I and mitochondrial cytochrome c oxidase subunit III) were conducted to characterize the coccidium or coccidia responsible for disease outbreaks in these ferrets in different age classes and years. Results suggest that a single Eimeria species, E. ictidea, was the cause of enteric coccidiosis in both SSP populations in both juvenile and adult age classes in all years evaluated. Wider research is indicated to determine whether these findings are representative of the broader captive and wild BFF populations.

Standardized ileal digestibility of amino acids in broiler chickens fed single or mixture of feed ingredients-based diets with or without Eimeria challenge.

The effect of Eimeria challenge on standardized ileal digestibility (SID) of amino acids (AA) in major poultry feed ingredients fed to broiler chickens was determined. A total of 840 male 9-day-old Ross 708 chicks were placed in 84 cages (10 birds/cage) and allocated to either a nitrogen-free diet (NFD) or one of the 6 test diets based on a single or mixture of feed ingredients as the sole source of AA (n = 12). Test diets were: 1) corn, 2) wheat, 3) soybean meal (SBM), 4) pork meal (PM), 5) corn, SBM, and PM (CSP), and 6) wheat, SBM, and PM (WSP). On d 10, birds in 6 cages/diet were orally gavaged with 1 mL of E. acervulina and E. maxima mixture and the other 6 cages with sham. On d 15, birds were bled for plasma AA and necropsied for intestinal lesion scores and ileal digesta samples. Challenge decreased (P < 0.05) plasma concentration of Arg, His, Thr, Asp, Gln, and Tyr and increased concentration of Lys, Ile, Leu, and Val. There was a diet by challenge interaction (P < 0.05) on intestinal lesion scores with birds fed mixed diets exhibiting more severe lesions than birds fed single ingredient diets. Diet by challenge interacted (P < 0.05) on ileal total endogenous flow (ITEF) of AA except for Arg, Met, Ala, Asp, and Cys, such that challenged birds fed the mixed, particularly WSP, had higher ITEF of AA compared to single ingredients birds. Diet and challenge interaction (P < 0.05) was observed for SID of Arg, Thr, Val, Glu, and Gly. Challenge decreased (P < 0.05) SID of most AA except for Met, Asp, and Cys with the largest impact seen on Lys, His, Ser, and Thr. With exception of Arg, Thr, Asp, and Cys, birds fed mixed diets had higher (P < 0.05) SID values compared to birds fed single ingredients. In conclusion, Eimeria reduced plasma availability and ileal digestibility of most AA. However, challenge interaction with diet composition on SID of some AA warrants further investigations.

Draft Hybrid Genome Assembly of a Canadian Cyclospora cayetanensis Isolate.

The apicomplexan parasite Cyclospora cayetanensis causes foodborne gastrointestinal disease in humans. Here, we report the first hybrid assembly for C. cayetanensis, which uses both Illumina MiSeq and Oxford Nanopore Technologies MinION platforms to generate genomic sequence data. The final genome assembly consists of 44,586,677 bases represented in 313 contigs.

Genotyping Canadian Cyclospora cayetanensis Isolates to Supplement Cyclosporiasis Outbreak Investigations.

Cyclospora cayetanensis is an emerging foodborne parasite that causes cyclosporiasis, an enteric disease of humans. Domestically acquired outbreaks have been reported in Canada every spring or summer since 2013. To date, investigations into the potential sources of infection have relied solely on epidemiological data. To supplement the epidemiological data with genetic information, we genotyped 169 Canadian cyclosporiasis cases from stool specimens collected from 2010 to 2021 using an existing eight-marker targeted amplicon deep (TADS) scheme specific to C. cayetanensis as previously described by the US Centers for Disease Control and Prevention (CDC). This is the first study to genotype Canadian Cyclospora cayetanensis isolates, and it focuses on evaluating the genotyping performance and genetic clustering. Genotyping information was successfully collected with at least part of one of the markers in the TADS assay for 97.9% of specimens, and 81.1% of cyclosporiasis cases met the minimum requirements to genetically cluster into 20 groups. The performance of the scheme suggests that examining cyclosporiasis cases genetically will be a valuable tool for supplementing epidemiological outbreak investigations and to minimize further infections. Further research is required to expand the number of discriminatory markers to improve genetic clustering.