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.

Multilocus Genotyping of Sympatric Hepatozoon Species Infecting the Blood of Ontario Ranid Frogs Reinforces Species Differentiation and Identifies an Unnamed Hepatozoon Species.

Intraerythrocytic gamonts of at least 2 named Hepatozoon species have been reported to infect the erythrocytes of ranid frogs in Ontario, Canada. Although gamonts of both species are morphometrically similar, the cytopathological changes that 1 of these species, Hepatozoon clamatae, causes to host erythrocytes, manifested by nuclear fragmentation, was used historically to distinguish this parasite from Hepatozoon catesbianae. Molecular characterization of these 2 Hepatozoon species has been equivocal in correlating genotype with gamont morphotype. Amplification and sequencing of multiple potential genotyping loci within the nuclear (18S ribosomal deoxyribonucleic acid [rDNA]; internal transcribed spacer 1), apicoplast (23S rDNA), and mitochondrial genomes (complete genomes, cytochrome c oxidase subunits I and III [COI and COIII], and cytochrome b) were conducted on Hepatozoon species that infect ranid frogs in Ontario. Sequence data were then used to evaluate the diversity of parasites present in these amphibian hosts and to assign genotypes to gamont morphotypes, if possible. Three distinct genotypes were identified at all loci; the data permitted the discovery of a third, formerly unrecognized Hepatozoon species in ranid frogs from Ontario. Although all genetic loci demonstrated differences between Hepatozoon species, mitochondrial COIII sequences were most suitable for genotypic differentiation of these parasites of frogs. Linking genotypes to gamont morphotypes proved impossible; genotypes identified as H. catesbianae and H. clamatae were found in infections with or without nuclear fragmentation of their host erythrocytes. This suggests that differentiating these species must rely on suitable genotyping methods for identification in the blood of their amphibian intermediate hosts.

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.

Babesia odocoilei and zoonotic pathogens identified from Ixodes scapularis ticks in southern Ontario, Canada.

Cervid babesiosis, caused by the protozoan hemoparasite Babesia odocoilei and transmitted by the blacklegged tick Ixodes scapularis, is an emerging disease of Canadian cervids. This pathogen has not yet been described in humans. Data are lacking on the role of migratory birds in the adventitious spread of Ba. odocoilei-infected ticks, as well as on the infection status of I. scapularis in environments used by susceptible wildlife hosts. Following a high-mortality outbreak of cervid babesiosis at the Toronto Zoo [TZ], the present study was initiated to investigate Ba. odocoilei and other tick-borne pathogens of veterinary and public health importance (Borrelia burgdorferi sensu stricto (s.s.), Anaplasma phagocytophilum, Borrelia miyamotoi, and Babesia microti) in I. scapularis at three sites in southern Ontario, Canada. Blanket dragging for questing ticks yielded I. scapularis from the three sites evaluated: TZ, Point Pelee National Park, and Long Point Bird Observatory [LPBO]. Babesia odocoilei was identified in I. scapularis collected by dragging at the TZ and at LPBO. Borrelia burgdorferi s.s. was identified in I. scapularis at all three sites. Anaplasma phagocytophilum was identified in I. scapularis collected from the TZ. During the springs of 2016 and 2017, 1102 northward-migrating birds were examined for ticks at LPBO. One or more I. scapularis were found on 3.2% of birds (n = 595) in 2016, and 6.7% (n = 507) of birds in 2017. Overall, across both years, 0.2% and 0.5% of birds carried one or more I. scapularis ticks that tested PCR-positive for Ba. odocoilei and Bo. burgdorferi s.s., respectively. These data indicate that Ba. odocoilei-positive I. scapularis are found in southern Ontario, and suggest that bird-borne ticks have the potential to contribute to range expansion of both Ba. odocoilei and Bo. burgdorferi s.s. in Canada.

Klossiella equi Infecting Kidneys of Ontario Horses: Life Cycle Features and Multilocus Sequence-Based Genotyping Confirm the Genus Klossiella Belongs In the Adeleorina (Apicomplexa: Coccidia).

Species in the genus Klossiella Smith and Johnson, 1902 are unique among the suborder Adeleorina because they are monoxenous in mammals exclusively, whereas all other reported members of the Adeleorina use invertebrates as definitive hosts. Unlike other coccidia, all members of the Adeleorina undergo syzygy, the association of microgamonts and macrogamonts before maturation to gametes and syngamy. After fertilization, many members of the Adeleorina produce thin-walled polysporocystic oocysts. Despite being biologically similar to other members of the Adeleorina, the phylogenetic placement of the genus Klossiella has been questioned based on its unique host affinity. In the present study, 2 cases of Klossiella equi were reported from the kidneys of horses in Ontario. Details of the life cycle as well as mitochondrial and nuclear 18S ribosomal DNA ( 18S rDNA) sequences were analyzed to provide both morphological and molecular evidence for the phylogenetic placement of K. equi. Initially, various stages of the life cycle were identified in histological slides prepared from the kidney tissue, and DNA was isolated from the infected tissue. Polymerase chain reaction and Sanger sequencing were used to generate a complete mitochondrial genome sequence (6,569 bp) and a partial 18S rDNA sequence (1,443 bp). The K. equi 18S rDNA sequence was aligned with various publicly available apicomplexan 18S rDNA sequences. This alignment was used to generate a phylogenetic tree based on Bayesian inference. Multiple K. equi stages were identified including meronts, microgamonts, and macrogamonts associating in syzygy as well as thin-walled oocysts in various stages of sporogonic development. The 18S rDNA sequence of K. equi positioned within the monophyletic Adeleorina clade. The mitochondrial genome of K. equi contained 3 coding sequences for cytochrome c oxidase I, cytochrome c oxidase III, and cytochrome b as well as various fragmented ribosomal sequences. These components were arranged in a unique order that has not been observed in other apicomplexan mitochondrial genomes sequenced to date. Overall, it was concluded that there were sufficient morphological and molecular data to confirm the placement of K. equi and the genus Klossiella among the Adeleorina. The biological and molecular data obtained from these cases may assist with future studies evaluating the prevalence and life history of this seemingly underreported parasite and better define the impact of K. equi on the health of domestic and wild equids.

Next generation sequencing from Hepatozoon canis (Apicomplexa: Coccidia: Adeleorina): Complete apicoplast genome and multiple mitochondrion-associated sequences.

Extrachromosomal genomes of the adeleorinid parasite Hepatozoon canis infecting an Israeli dog were investigated using next-generation and standard sequencing technologies. A complete apicoplast genome and several mitochondrion-associated sequences were generated. The apicoplast genome (31,869 bp) possessed two copies of both large subunit (23S) and small subunit (16S) ribosomal RNA genes (rDNA) within an inverted repeat region, as well as 22 protein-coding sequences, 25 transfer RNA genes (tDNA) and seven open reading frames of unknown function. Although circular-mapping, the apicoplast genome was physically linear according to next-generation data. Unlike other apicoplast genomes, genes encoding ribosomal protein S19 and tDNAs for alanine, aspartic acid, histidine, threonine and valine were not identified. No complete mitochondrial genome was recovered using next-generation data or directed PCR amplifications. Eight mitochondrion-associated (215-3523 bp) contigs assembled from next-generation data encoded a complete cytochrome c oxidase subunit I coding sequence, a complete cytochrome c oxidase subunit III coding sequence, two complete cytochrome B coding sequences, a non-coding, pseudogene for cytochrome B and multiple fragmented mitochondrial rDNA genes (SSUA, SSUB, SSUD, LSUC, LSUG, RNA6, RNA10, RNA14, RNA18). The paucity of NGS reads generating each of the mitochondrion-like sequences suggested that a complete mitochondrial genome at typically high copy number was absent in H. canis. In contrast, the complete nuclear rDNA unit sequence of H. canis (18S rDNA to 28S rDNA, 6977 bp) had >1000-fold next-generation coverage. Multiple divergent (from 93.6% to 99.9% pairwise identities) nuclear 18S rDNA contigs were generated (three types with 10 subtypes total). To our knowledge this is the first apicoplast genome sequenced from any adeleorinid coccidium and the first mitochondrion-associated sequences from this serious pathogen of wild and domestic canids. These newly generated sequences may provide useful genetic loci for high-resolution species-level genotyping that is currently impossible using existing nuclear rDNA targets.

MOLECULAR DETECTION OF BABESIA ODOCOILEI IN WILD, FARMED, AND ZOO CERVIDS IN ONTARIO, CANADA.

Babesia odocoilei, a tick-borne protozoan hemoparasite of white-tailed deer ( Odocoileus virginianus), is being increasingly recognized as a cause of disease in captive cervids in North America. Historically endemic in white-tailed deer, the natural wildlife reservoir in the southeastern US, B. odocoilei has been recently associated with hemolytic anemia in captive Eurasian tundra reindeer ( Rangifer tarandus tarandus), wapiti ( Cervus canadensis), and woodland caribou ( Rangifer tarandus caribou) in the northcentral and northeastern US and several Canadian provinces. The emergence of B. odocoilei is likely related to the northward expansion of the range of the tick vector, Ixodes scapularis, and possibly to cervid translocations. Following a disease outbreak in reindeer and wapiti at the Toronto Zoo in Ontario, Canada, we utilized a prospective postmortem survey to investigate the prevalence of B. odocoilei in wild, farmed, and zoo cervids in Ontario ( n=270) in 2016-18 by PCR and DNA sequencing of spleen samples. Zoo bovids have been suggested as potential hosts of B. odocoilei in zoos affected by cervid babesiosis, so we also collected postmortem samples from five species of bovids ( n=7) at the Toronto Zoo that died or were euthanized during this time. We detected B. odocoilei in 1% (2/142) of farmed red deer ( Cervus elaphus) as well as in 3% (1/29) of captive wapiti and 4% (3/68) of wild white-tailed deer. Tissues from all zoo bovids and caribou, zoo and wild moose ( Alces alces), and farmed white-tailed deer, wapiti-red deer hybrids, and fallow deer ( Dama dama), tested negative for B. odocoilei. No clinical cases of babesiosis were encountered during this study. These findings suggest that white-tailed deer are a potential natural wildlife reservoir for B. odocoilei in Ontario and that red deer and wapiti could serve as more-localized reservoirs.

Prevalence, Distribution, and Risk Factors Associated With Macracanthorhynchus ingens Infections In Raccoons From Ontario, Canada.

Macracanthorhynchus ingens is an acanthocephalan parasite commonly found in raccoons ( Procyon lotor) in the United States. Little is known, however, about the prevalence and distribution of M. ingens in raccoons in Canada. Our objective was to investigate the prevalence, distribution, and risk factors associated with M. ingens infection in raccoons in southern Ontario, Canada. Raccoon carcasses submitted to the Ontario/Nunavut region of the Canadian Wildlife Health Cooperative for post mortem from June 2016 to January 2017 (n = 380) were examined for the presence of gastrointestinal helminths. Macracanthorhynchus ingens was found in raccoons from areas across southern Ontario where carcasses were submitted. The prevalence of M. ingens in our sample was 14.0% (95% CI = 10.6-17.8) with a median of 4 worms per infected host (range 1-46). Univariable logistic regression modeling was conducted to examine the influence of age, sex, season, degree of urbanization (urban/suburban/rural), and Baylisascaris procyonis infection on the presence of M. ingens. Significant associations were found between M. ingens infection and degree of urbanization as well as B. procyonis infection. No associations were found between M. ingens infection and age, sex, or season. To our knowledge, this is the first report describing the prevalence and distribution of M. ingens in raccoons in Canada.

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).

The complete mitochondrial genome sequence of Hepatozoon catesbianae (Apicomplexa: Coccidia: Adeleorina), a blood parasite of the green frog, Lithobates (formerly Rana) clamitans.

A complete mitochondrial genome for the blood parasite Hepatozoon catesbianae (Alveolata; Apicomplexa; Coccidia; Adeleorina; Hepatozoidae) was obtained through PCR amplification and direct sequencing of resulting PCR products. The mitochondrial genome of H. catesbianae is 6,397 bp in length and contains 3 protein-coding genes (cytochrome c oxidase subunit I [COI]; cytochrome c oxidase subunit III [COIII]; and cytochrome B [CytB]). Sequence similarities to previously published mitochondrial genomes of other apicomplexan parasites permitted annotation of 23 putative rDNA fragments in the mitochondrial genome of H. catesbianae, 14 large subunit rDNA fragments, and 9 small subunit rDNA fragments. Sequences corresponding to rDNA fragments RNA5, RNA8, RNA11, and RNA19 of Plasmodium falciparum were not identified in the mitrochondrial genome sequence of H. catesbianae. Although the presence of 3 protein-coding regions and numerous putative rDNA fragments is a feature typical for apicomplexan mitochondrial genomes, the mitochondrial genome of H. catesbianae possesses a structure and gene organization that is distinct among the Apicomplexa. This is the first complete mitochondrial genome sequence obtained from any apicomplexan parasite in the suborder Adeleorina.