The seroprevalence and risk factors for exposure to Neospora caninum and Neospora hughesi in Ontario broodmares.

The seroprevalence and risk factors for exposure to Neospora caninum and Neospora hughesi in broodmares in Ontario were investigated. Sixty of the 219 (27.4%) study broodmares were seropositive for N. caninum and 65/219 (29.7%) for N. hughesi with cut-offs of ≥1:40 and ≥1:160, respectively. Thirty-one of 63 participating farms (49.2%) had at least 1 broodmare seropositive for N. caninum. Thirty-three of the 63 (52.4%) participating farms had at least 1 broodmare positive for N. hughesi. Risk factors for N. caninum included presence of farm dogs (OR = 6.70; 95% CI = 2.14-20.97; p = 0.001), and high stocking density (OR = 2.83; 95% CI = 1.27-6.30; p = 0.011). Presence of livestock, excluding cattle, was associated with reduced risk of exposure (OR = 0.17; 95% CI = 0.06-0.53; p = 0.002). The only risk factor for exposure to N. hughesi was feeding hay on the ground in the paddock (OR = 4.31; 95% CI = 1.65-11.22; p = 0.003). This study demonstrated widespread exposure to Neospora spp. in broodmares in Ontario.

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.

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.

Research Note: Isolation, speciation, and anticoccidial sensitivity of Eimeria spp. recovered from wild turkey feces in the United States.

Between 2018 and 2020, over 100 wild turkey fecal samples were collected from the Eastern and Central thirds of the United States, where commercial turkey production is uncommon. We hypothesized that anticoccidial-sensitive Eimeria spp. would be present in wild turkey fecal samples. Samples containing Eimeria spp. oocysts were amplified in vivo. If propagation was successful, the samples were PCR-speciated and subjected to anticoccidial sensitivity testing (AST) for key members of both ionophore and chemical categories of anticoccidial drugs. The purpose of this study was to isolate Eimeria spp. relevant to commercial turkey production that possessed sensitivity to monensin, zoalene, and amprolium. Future research would evaluate the efficacy of wild turkey Eimeria spp. as vaccine candidates for reducing coccidiosis in commercial turkey flocks utilizing single oocyst-derived stocks obtained in the present study.

Impact of Eimeria meleagrimitis and intermittent amprolium treatment on performance and the gut microbiome composition of Turkey poults.

Introduction: Drug-sensitive live coccidiosis vaccines have been used to control coccidiosis and renew drug sensitivity in commercial chicken operations. However, only limited species coverage vaccines have been available for commercial turkey producers. This study aimed to assess the effect of an E. meleagrimitis vaccine candidate, with and without amprolium intervention, on performance and oocyst shedding. Additionally, the effect of vaccination, amprolium treatment, and E. meleagrimitis challenge on intestinal integrity and microbiome composition was evaluated. Methods: Experimental groups included: (1) NC (non-vaccinated, non-challenged control); (2) PC (non-vaccinated, challenged control); (3) VX + Amprol (E. meleagrimitis candidate vaccine + amprolium); and 4) VX (E. meleagrimitis candidate vaccine). For VX groups, 50% of the direct poults were orally vaccinated at DOH with 50 sporulated E. meleagrimitis oocysts and were comingled with contact or non-vaccinated poults for the duration of the study. From d10-14, VX + Amprol group received amprolium (0.024%) in the drinking water. All groups except NC were orally challenged with 95K E. meleagrimitis sporulated oocysts/mL/poult at d23. At d29, ileal and cecal contents were collected for 16S rRNA gene-based microbiome analysis. Results and Discussion: VX did not affect performance during the pre-challenge period. At d23-29 (post-challenge), VX groups had significantly (P < 0.05) higher BWG than the PC group. Contacts and directs of VX groups in LS had significantly reduced compared to PC. As anticipated, amprolium treatment markedly reduced fecal and litter OPG for the VX + Amprol group compared to the VX group which did not receive amprolium. The ileal and cecal content results showed that the PC group had different bacterial diversity and structure, including alpha and beta diversity, compared to NC. Linear discriminant analysis Effect Size (LEfSe) identified that Lactobacillus salivarius (ASV2) was enriched in PC's ileal and cecal content. Compared to NC and PC, the vaccinated groups showed no distinct clusters, but there were similarities in the ileal and cecal communities based on Bray-Curtis and Jaccard distances. In conclusion, these results indicate that vaccination with this strain of E. meleagrimitis, with or without amprolium intervention, caused a very mild infection that induced protective immunity and challenge markedly affected both the ileal and cecal microbiome.

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.

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.

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.

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.

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.

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.

The complete mitochondrial genome sequence of Eimeria leuckarti (Eimeriidae, Coccidia, Apicomplexa) infecting domestic horses (Equus ferus caballus).

The complete mitochondrial genome of Eimeria leuckarti (Eimeriidae, Coccidia, Apicomplexa) was obtained. This morphologically distinctive coccidium is considered to be the only valid Eimeria species of equids and it infects a range of both domestic and wild horses and their relatives. Despite the distinctive appearance of the oocysts of E. leuckarti, the mitochondrial genome organization and gene contents were comparable to other Eimeria spp. and related eimeriid coccidia infecting a range of mammals and birds. The greatly reduced 6242 bp genome is circular-mapping and contains three protein-coding genes (COI, COIII, CytB), 18 fragments encoding the large subunit rRNA (LSU), and 13 fragments encoding the small subunit (SSU) rRNA. No tRNA was encoded similar to other Apicomplexa. A Bayesian inference tree based on aligned CDS and rDNA fragments from Eimeria leuckarti and 34 other coccidia demonstrated that this mt genome has close phylogenetic affinities to Eimeria and Isospora species, and related eimeriid coccidia.

DESCRIPTION OF A NEW EIMERIA SPECIES (APICOMPLEXA: EIMERIIDAE) RESPONSIBLE FOR CLINICAL COCCIDIOSIS IN COMMERCIAL CHUKAR PARTRIDGE (ALECTORIS CHUKAR).

Recurrent coccidiosis affecting a commercial chukar partridge (Alectoris chukar) farm in Ontario, Canada was investigated. The responsible pathogenic Eimeria species was isolated for biological characterization. The uniformity of oocyst morphometrics supported that only a single Eimeria sp. was present. Experimental infections with coccidia-free chukars were used to describe exogenous and endogenous developmental stages of the parasite. The prepatent period of the causative Eimeria species was 5 days and patency lasted 11 days; fecundity was 1,573 to 30,057, with the highest fecundity recorded with the lowest challenge dose. Endogenous development was elucidated histologically from samples collected at 8 locations along the intestinal tract at 26 time points throughout prepatency. The parasite had 5 asexual generations before oocyst formation that were located from the mid-jejunum to the mid-rectum and in the ceca. Sporulation of oocysts suspended in potassium dichromate at room temperature (22 C) occurred within 24 hr. Oocysts (n = 50) averaged 21.8 by 18.6 µm and featured a polar granule; sporocysts (n = 50) averaged 10.9 by 7.1 µm and possessed a Stieda body, sub-Stieda body, sporozoite refractile bodies, and sporocyst residuum. Comparisons with described Eimeria spp. infecting partridges suggest that the biological features of this pathogenic species are unique; similarly, sequences from both mitochondrial and nuclear loci support the naming of this new Eimeria species.

DESCRIPTION OF THE FIRST KLOSSIA SPECIES (APICOMPLEXA: EUCOCCIDIORIDA: ADELEORINA: ADELEIDAE) INFECTING A PULMONATE LAND SNAIL, TRIODOPSIS HOPETONENSIS (MOLLUSCA: POLYGYRIDAE), IN NORTH AMERICA.

Snails identified as Triodopsis hopetonensis were collected (n = 18) from the University of Arkansas-Fayetteville campus in December 2018. Additional snails were collected in April 2019 (n = 9) and in September 2019 (n = 9). Kidney tissues were examined using light microscopy, and polysporocystic oocysts were observed. Sporulated oocysts (n = 2) measured 78 ± 3.4 µm × 76 ± 2.9 µm with an irregular oocyst residuum and contained an estimated 44-55 tetrazoic sporocysts. The sporocysts (n = 10) measured 13 ± 0.5 µm × 11 ± 1.5 µm with an indistinct, irregularly placed, sporocyst residuum and usually contained 4 sporozoites, although an octozoic variant was observed. DNA was extracted from the snail kidney tissues and used as a template for polymerase chain reaction (PCR). PCR was used to determine the infection status of the snails; 13 of 36 (36%) specimens were found to be infected with a new Klossia species, and only 3 (8%) of these infections were detected using light microscopy. The complete nuclear 18S ribosomal DNA (1,800 bp) and mitochondrial genomes (6,775 bp) were generated, and they differentiated this parasite from the type species Klossia helicina and support the description of this new Klossia species, Klossia razorbacki n. sp. This is the first Klossia species to be described from any North American snail.

Extended-spectrum cephalosporin resistance in Escherichia coli from broiler chickens raised with or without antibiotics in Ontario, Canada.

Extended-spectrum cephalosporin (ESC)-resistant Escherichia coli were isolated from fecal samples enriched in ESC-containing broth over two years from broiler chickens raised with and without therapeutic antimicrobials. Pooled samples were obtained from 13 different farms in Canada over two rearing cycles each. Resistant isolates were screened for blaCMY, blaCTX-M, and blaSHV by PCR, and several isolates were sequenced and assembled using Oxford Nanopore MinION technology. Flocks raised with or without antimicrobials contained similar ESC-resistant E. coli. Some plasmids were found in isolates from both farm types and many shared replicon types (IncI1, F, C, and B/O/K/Z) and resistance determinants. Although the use of cephalosporins has stopped in poultry production in Canada, the prevalence of ESC-resistant E. coli found in this study remained high; therefore, further studies are required to determine routes of transmission and persistence.

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.

Molecular characterization of enteric coccidia from domestic ferrets (Mustela putorius furo).

Combined morphometric and molecular characterization of coccidia that infect domestic ferrets (Mustela putorius furo) was completed to improve the diagnostic specificity of 'coccidiosis' in this host. Coccidia-positive fecal samples (n = 11) and formalin-fixed paraffin-embedded intestinal tissues (n = 3) from domestic ferrets were collected from diagnostic laboratories in Canada and Europe. An average of 3.5 and 13 domestic ferret fecal samples per year were coccidia-positive when tested by Canadian and European diagnostic laboratories, respectively, during the period 2008-2015. Oocyst morphometrics and sequence genotyping at two loci (nuclear 18S rDNA [nu 18S] and mitochondrial cytochrome c oxidase subunit I [mt COI]) were conducted on all samples. The first nu 18S and mt COI sequences for Cystoisospora laidlawi, and the first mt COI sequence for Eimeria furonis were generated during this study. Phylogenetic analysis of the mitochondrial COI sequences demonstrated that E. furonis was most closely related to E. ictidea isolated from a black-footed ferret (Mustela nigripes) and that C. laidlawi was closely related to C. canis and C. felis. The identifications provided by diagnostic laboratories of the specific parasite species present in a sample showed poor agreement with their identifications based on genotyping obtained in this study. Molecular techniques appear to be essential for determining the specific coccidial species responsible for individual and group outbreaks of coccidiosis and for further understanding of eimeriid host-parasite relationships.

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.