A novel Bacillus sp. with rapid growth property and high enzyme activity that allows efficient fermentation of soybean meal for improving digestibility in growing pigs.

AIMS: Soybean meal (SBM) contributes high-quality dietary protein for pigs. However, it also contains antinutritional factors such as allergenic high molecular weight proteins and non-starch polysaccharides (NSP) that limit its use. Therefore, the objective of this study was to screen and characterize a robust Bacillus sp. from camel dung for soybean meal fermentation to improve the digestibility in growing pigs. METHODS AND RESULTS: Molecular characterization revealed that isolate 9 (hereinafter referred to as "CP-9") was a Bacillus subtilis strain. It secreted cellulase (0.07 U ml-1 ), xylanase (1.91 U ml-1 ), and amylase (2.66 U ml-1 ) into the culture supernatant. Isolate CP-9 showed rapid growth on LB agar plates and grew at a wide range of pH (3.0-9.0) and temperatures (23-50°C) in LB broth. Protein profiling of SBM using SDS-PAGE showed a significant reduction of large globular proteins to small peptides after 48 h of fermentation. On a dry matter basis, neutral detergent fibre (NDF) of the fermented SBM (F-SBM) was decreased by 34.25% (from 9.72 to 7.24%) with an increase in CP content by 16.54% (from 48.74 to 56.80%). Pigs fed with a semi-purified diet formulated with F-SBM as the sole source of crude protein had higher (p < 0.05) apparent ileal digestibility (AID) of DM (80.0 vs. 71.7%), ash (55.6 vs. 36.1%), CP (84.2 vs. 78.3%), NDF (70.9 vs. 66.0%), and ADF (62.4 vs. 53.3%) compared with pigs fed with unfermented soybean meal (UF-SBM). CONCLUSIONS: A novel Bacillus subtilis strain CP-9 was isolated and characterized from camel dung for efficient fermentation of SBM. This bacterium ameliorates physico-chemical characteristics of F-SBM and improved nutrient digestibility in growing pigs. SIGNIFICANCE AND IMPACT OF THE STUDY: Our data suggest that a low-cost solid-state SBM fermentation was developed using this newly isolated bacterium. The resultant F-SBM improved the nutrient digestibility in growing pigs.

Neurologic Baylisascaris procyonis infection in a young dog.

A 14-week-old female Boston terrier-cross dog with intermittent gastroenteritis and an eosinophilia developed progressive neurologic disease with ataxia progressing to uncontrolled paddling. Autopsy revealed Baylisascaris procyonis larvae in 4 of 7 brain sections, with severe eosinophilic meningoencephalitis. Diagnosis was confirmed with polymerase chain reaction (PCR) and DNA sequencing tests of fresh and paraffin-embedded brain in conjunction with the compatible histologic appearance.

Infection neurologique à Baylisascaris procyonis chez une jeune chienne. Une jeune chienne terrier de Boston de race croisée âgée de 14 semaines a été atteinte de gastroentérite intermittente et d'éosinophilie et a développé une maladie neurologique progressive avec de l'ataxie progressant à des mouvements involontaires. L'autopsie a révélé une larve de Baylisascaris procyonis dans 4 des 7 sections du cerveau, avec une méningo-encéphalite éosinophilique grave. Le diagnostic a été confirmé par amplification en chaîne par polymérase (PCR) et des tests de séquençage de l'ADN de tissus du cerveau frais et inclus dans la paraffine conjointement à l'apparence histologique compatible.(Traduit par Isabelle Vallières).

SYSTEMIC ENCEPHALITOZOONOSIS DUE TO ENCEPHALITOZOON CUNICULI STRAIN IV IN A VANCOUVER ISLAND MARMOT ( MARMOTA VANCOUVERENSIS).

A 2-mo-old Vancouver Island marmot ( Marmota vancouverensis), housed at a quarantined breeding facility, presented for acute obtundation and vestibular ataxia. Physical examination revealed poor growth compared with littermates, poor nutritional condition, and mild dehydration. The animal's condition deteriorated over 24 hr, and it was euthanized following the development of generalized seizures. No gross abnormalities were observed upon postmortem evaluation. Histologic evaluation revealed severe, multifocal, granulomatous and lymphoplasmacytic meningoencephalomyelitis and interstitial nephritis, with intralesional, intracytoplasmic spore-filled, parasitophorous vacuoles and segmental, multi-organ, fibrinoid vasculitis (disseminated encephalitozoonosis). The etiologic agent was evident by hematoxylin and eosin and Gram-chromotrope stains, and confirmed as Encephalitozoon cuniculi by polymerase chain reaction on brain tissue. Sequencing of the internal transcribed spacer region of the rRNA gene showed 100% homology with E. cuniculi strain IV, which is a newly described genotype. This is the first report of encephalitozoonosis in this critically endangered species.

ECHINOCOCCUS EQUINUS HYDATID CYST IN THE LIVER OF A PRZEWALSKI'S HORSE ( EQUUS PRZEWALSKII) IN A CANADIAN ZOO.

A 23-yr-old captive-born Przewalski's horse mare ( Equus przewalskii) was euthanized at a Canadian zoo because of severe colic resulting from rupture of a jejunal pseudodiverticulum. An incidental finding of an encysted larval cestode within a hepatic granuloma was diagnosed on histopathology. Gel-based polymerase chain reaction (PCR) on liver tissue was positive for Echinococcus granulosus sensu lato, and deoxyribonucleic acid sequencing of the PCR product was 100% homologous with Echinococcus equinus. This appears to be the first molecular confirmation of E. equinus in North America, and the first report of cystic echinococcosis in a Przewalski's horse.

Complete genome sequences of Mycoplasma cynos and Mycoplasma felis isolated from dogs and cats with infectious respiratory disease.

Mycoplasma cynos is a primary agent of pneumonia in dogs, and Mycoplasma felis is associated with upper respiratory tract disease in cats. We present complete genome sequences of 26 isolates from clinically affected dogs and cats. These genome sequences will facilitate new molecular and epidemiological analyses.

Additional diagnoses of Echinococcus multilocularis in eastern chipmunks (Tamias striatus) from southern Ontario - results from ongoing surveillance for E. multilocularis in intermediate hosts in Ontario, Canada.

Echinococcus multilocularis, a cestode with zoonotic potential, is now known to have a high prevalence in wild canid definitive hosts of southern Ontario. The distribution of E. multilocularis across this region in red foxes (Vulpes vulpes) and coyotes (Canis latrans) is widespread yet heterogenous. In contrast, confirmed diagnoses of E. multilocularis in wild free-ranging intermediate hosts within Ontario are currently limited to a single eastern chipmunk (Tamias striatus). These findings prompted ongoing surveillance efforts in intermediate host species, primarily rodents. Our report describes the results of passive surveillance through wildlife carcass submissions to the Canadian Wildlife Health Cooperative (CWHC) and targeted active sampling of small mammal species from 2018 to 2023; a second and third eastern chipmunk were found to be infected with E. multilocularis. However, these were the only occurrences from surveillance efforts which collectively totaled 510 rodents and other small mammals. Continued surveillance for E. multilocularis in intermediate hosts is of high importance in light of the recent emergence of this parasite in Ontario.

Paranannizziopsis spp. infections in wild snakes and a qPCR assay for detection of the fungus.

The emergence of ophidiomycosis (or snake fungal disease) in snakes has prompted increased awareness of the potential effects of fungal infections on wild reptile populations. Yet, aside from Ophidiomyces ophidiicola, little is known about other mycoses affecting wild reptiles. The closely related genus Paranannizziopsis has been associated with dermatomycosis in snakes and tuataras in captive collections, and P. australasiensis was recently identified as the cause of skin infections in non-native wild panther chameleons (Furcifer pardalis) in Florida, USA. Here we describe five cases of Paranannizziopsis spp. associated with skin lesions in wild snakes in North America and one additional case from a captive snake from Connecticut, USA. In addition to demonstrating that wild Nearctic snakes can serve as a host for these fungi, we also provide evidence that the genus Paranannizziopsis is widespread in wild snakes, with cases being identified in Louisiana (USA), Minnesota (USA), Virginia (USA), and British Columbia (Canada). Phylogenetic analyses conducted on multiple loci of the fungal strains we isolated identified P. australasiensis in Louisiana and Virginia; the remaining strains from Minnesota and British Columbia did not cluster with any of the described species of Paranannizziopsis, although the strains from British Columbia appear to represent a single lineage. Finally, we designed a pan-Paranannizziopsis real-time PCR assay targeting the internal transcribed spacer region 2. This assay successfully detected DNA of all described species of Paranannizziopsis and the two potentially novel taxa isolated in this study and did not cross-react with closely related fungi or other fungi commonly found on the skin of snakes. The assay was 100% sensitive and specific when screening clinical (skin tissue or skin swab) samples, although full determination of the assay's performance will require additional follow up due to the small number of clinical samples (n = 14 from 11 snakes) available for testing in our study. Nonetheless, the PCR assay can provide an important tool in further investigating the prevalence, distribution, and host range of Paranannizziopsis spp. and facilitate more rapid diagnosis of Paranannizziopsis spp. infections that are otherwise difficult to differentiate from other dermatomycoses.

Widespread occurrence of Batrachochytrium dendrobatidis in Ontario, Canada, and predicted habitat suitability for the emerging Batrachochytrium salamandrivorans.

Chytridiomycosis, caused by the fungi Batrachochytrium dendrobatidis and Batrachochytrium salamandrivorans, is associated with massive amphibian mortality events worldwide and with some species' extinctions. Previous ecological niche models suggest that B. dendrobatidis is not well-suited to northern, temperate climates, but these predictions have often relied on datasets in which northern latitudes are underrepresented. Recent northern detections of B. dendrobatidis suggest that these models may have underestimated the suitability of higher latitudes for this fungus. We used qPCR to test for B. dendrobatidis in 1,041 non-invasive epithelial swab samples from 18 species of amphibians collected across 735,345 km2 in Ontario and Akimiski Island (Nunavut), Canada. We detected the pathogen in 113 samples (10.9%) from 11 species. Only one specimen exhibited potential clinical signs of disease. We used these data to produce six Species Distribution Models of B. dendrobatidis, which classified half of the study area as potential habitat for the fungus. We also tested each sample for B. salamandrivorans, an emerging pathogen that is causing alarming declines in European salamanders, but is not yet detected in North America. We did not detect B. salamandrivorans in any of the samples, providing a baseline for future surveillance. We assessed the potential risk of future introduction by comparing salamander richness to temperature-dependent mortality, predicted by a previous exposure study. Areas with the highest species diversity and predicted mortality risk extended 60,530 km2 across southern Ontario, highlighting the potential threat B. salamandrivorans poses to northern Nearctic amphibians. Preventing initial introduction will require coordinated, transboundary regulation of trade in amphibians (including frogs that can carry and disperse B. salamandrivorans), and surveillance of the pathways of introduction (e.g., water and wildlife). Our results can inform surveillance for both pathogens and efforts to mitigate the spread of chytridiomycosis through wild populations.

Evaluation of the zoonotic potential of Giardia duodenalis in fecal samples from dogs and cats in Ontario.

This study determined the distribution and zoonotic potential of Giardia duodenalis assemblage types among canine and feline fecal samples from Ontario. The effectiveness of Giardia assemblage typing methods by sequencing the genes of small subunit ribosomal RNA (ssu-rRNA), ß-giardin (bg), glutamate dehydrogenase (gdh), and triose phosphate isomerase (tpi) was evaluated simultaneously. From 2008 to 2010, 118 canine and 15 feline Giardia positive fecal samples were tested. The ssu-rRNA sequencing method typed 64% (75/118) and 87% (13/15) of the Giardia-positive canine and feline samples, respectively. Among the typeable samples, 68% (51/75) of canine samples contained G. duodenalis assemblage D and 31% (23/75) contained G. duodenalis assemblage C (both non-zoonotic assemblage types). Only 1% (1/75) of the typeable canine samples contained a potentially zoonotic assemblage B. In contrast, 100% (13/13) of the typeable feline samples contained potentially zoonotic assemblages A (n = 12) or B (n = 1).

SARS-CoV2 spike protein gene variants with N501T and G142D mutation-dominated infections in mink in the United States.

Large numbers of mink have been infected with SARS-CoV2 containing the spike protein Y453F mutation in Europe, causing zoonosis concerns. To evaluate the genetic characteristics of the U.S. and Canadian mink-derived SARS-CoV2 sequences, we analyzed all animal-derived (977) and all Canadian (19,529) and U.S. (173,277) SARS-CoV2 sequences deposited in GISAID from December 2019 to March 12, 2021, and identified 2 dominant novel variants, the N501T-G142D variant and N501T-G142D-F486L variant, in the U.S. mink-derived SARS-CoV2 sequences. These variants were not found in mink from Canada or other countries. The Y453F mutation was not identified in the mink-derived sequences in the United States and Canada. The N501T mutation occurred 2 mo earlier in humans than in mink in the United States, and the novel N501T-G142D and N501T-G142D-F486L variants were found in humans prior to mink. Our results suggest that the novel SARS-CoV2 variants may have evolved during human infection and were then transmitted to mink populations in the United States.

Prevalence of Mycoplasma synoviae and Its Impact on Productivity in Commercial Poultry Farms in Quebec, Canada.

Mycoplasma synoviae (MS) is associated with upper respiratory disease, joint, and reproductive system disease in poultry. Economic losses are due to stunting, increased mortality, lower egg production, and higher slaughterhouse condemnations. The seroprevalence of MS is increasing worldwide, and more pathogenic strains have emerged over the past few years. Where this increase is noted, the economic consequences are considerable, even when there are no obvious clinical signs. The best control strategy is to maintain mycoplasma-free flocks. Since 2014 in Quebec, Canada, MS has been isolated with greater frequency in poultry farms and at times, as a primary pathogenic agent. The aim of this study was to evaluate the prevalence and impact of MS in commercial poultry farms in Quebec because the poultry industry was considering an insurance program that would cover losses in case of an outbreak. MS was shown to be present in all types of commercial production, although egg layers were principally affected with over 50% of flocks sampled being MS-positive in all producing regions of the province. On the basis of vlhA gene sequencing, several strains were identified with the most prevalent ones being type E, followed by Qc-1, a strain specific to Quebec. On average, the impact of MS on production parameters were not significant for any of the different types of commercial poultry production.

Prevalencia de Mycoplasma synoviae y su impacto en la productividad en granjas avícolas comerciales en Quebec, Canadá. Mycoplasma synoviae (MS) se asocia con enfermedades de las vías respiratorias superiores, problemas articulares y del sistema reproductivo en la avicultura comercial. Las pérdidas económicas se deben al retraso en el crecimiento, aumento de la mortalidad, menor producción de huevo y mayores decomisos en plantas de procesamiento. La seroprevalencia de M. synoviae está aumentando en todo el mundo y en los últimos años han surgido cepas más patógenas. En los lugares donde se ha observado este aumento, las consecuencias económicas han sido considerables, incluso cuando no se presentan signos clínicos evidentes. La mejor estrategia de control es mantener las parvadas libres de micoplasmas. Desde 2014 en Quebec, Canadá, M. synoviae se ha aislado con mayor frecuencia en granjas avícolas y en ocasiones como agente patógeno primario. El objetivo de este estudio fue evaluar la prevalencia y el impacto de M. synoviae en granjas avícolas comerciales en Quebec porque la industria avícola estaba considerando un programa de seguros que cubriría las pérdidas en caso de un brote. Se demostró que M. synoviae estaba presente en todos los tipos de producción comercial, aunque las aves de postura de huevo se vieron afectadas principalmente, con más del 50% de las parvadas muestreadas positivas a M. synoviae en todas las regiones productoras de la provincia. Con base en la secuenciación del gene vlhA, se identificaron varias cepas, siendo las más prevalentes el tipo E, seguidas de la cepa QC-1, que es específica de Quebec. En promedio, el impacto de M. synoviae en los parámetros de producción no fue significativo para ninguno de los diferentes tipos de producción avícola comerciales.

Effects of inflammatory stimuli on responses of macrophages to Mycoplasma bovis infection.

Inflammation in the respiratory tract is thought to worsen the disease response to Mycoplasma bovis infection. This study investigated the cells involved in this response with a focus on proteases and cytokines as harmful effector mechanisms. By immunohistochemistry, Mac387-positive macrophages were the main cell type comprising the foci of caseous necrosis in cattle with M. bovis pneumonia. Thus, the study evaluated how priming of different types of macrophages with bacterial lysate (or pro-inflammatory cytokines induced by the bacterial lysate) affected their responses to M. bovis infection. Inducible responses were detected in monocyte-derived macrophages (M1-MDMs and M2-MDMs), whereas pulmonary alveolar macrophages (PAMs) were minimally affected by priming or infection. M. bovis-infected MDMs secreted MMP-12 and SPLA2, and priming with pro-inflammatory cytokines increased the secretion of cathepsin B in response to M. bovis infection. Of these, there were higher concentrations of cathepsin B and SPLA2 in lungs with M. bovis pneumonia compared to healthy lungs, and these are potential mechanisms for macrophage-induced lung damage in M. bovis infection. Priming of MDMs with either bacterial lysate or with pro-inflammatory cytokines caused an enhanced response to M. bovis infection with respect to IL-8 and IL-1ß secretion. The findings of this study suggest proteases, lipases and cytokines derived from monocyte-derived macrophages as possible mediators by which prior inflammation in the respiratory tract worsen disease outcomes from M. bovis infection.

Identification of Antimicrobial Resistance-Associated Genes through Whole Genome Sequencing of Mycoplasma bovis Isolates with Different Antimicrobial Resistances.

Antimicrobial resistance (AMR) in Mycoplasma bovis has been previously associated with topoisomerase and ribosomal gene mutations rather than specific resistance-conferring genes. Using whole genome sequencing (WGS) to identify potential new AMR mechanisms for M. bovis, it was found that a 2019 clinical isolate with high MIC (2019-043682) for fluoroquinolones, macrolides, lincosamides, pleuromutilins and tetracyclines had a new core genome multilocus sequencing (cgMLST) type (ST10-like) and 91% sequence similarity to the published genome of M. bovis PG45. Closely related to PG45, a 1982 isolate (1982-M6152) shared the same cgMLST type (ST17), 97.2% sequence similarity and low MIC results. Known and potential AMR- associated genetic events were identified through multiple sequence alignment of the three genomes. Isolate 2019-043682 had 507 genes with non-synonymous mutations (NSMs) and 67 genes disrupted. Isolate 1982-M6152 had 81 NSMs and 20 disruptions. Using functional roles and known mechanisms of antimicrobials, a 55 gene subset was assessed for AMR potential. Seventeen were previously identified from other bacteria as sites of AMR mutation, 38 shared similar functions to them, and 11 contained gene-disrupting mutations. This study indicated that M. bovis may obtain high AMR characteristics by mutating or disrupting other functional genes, in addition to topoisomerases and ribosomal genes.

Diversity of Mycoplasma hyopneumoniae strains from pigs across Ontario, Canada.

Mycoplasma hyopneumoniae causes highly contagious swine enzootic pneumonia worldwide. It has been reported that highly diversified M. hyopneumoniae strains exist in different parts of the world. We found p146 gene sequencing analysis, an affordable and simple-to-perform typing method, to be specific and highly sensitive when applied to the molecular typing of 113 M. hyopneumoniae-positive clinical samples directly without culture. The samples were submitted to the Animal Health Laboratory at the University of Guelph (Ontario, Canada) during 2009-2017 from 40 different geographic areas in Ontario. Using a previously described criterion of grouping strains with < 4-bp differences into the same molecular type (p146 type), the 113 clinical samples clustered into 19 p146 genotypes. Dominant types were found in 2016 and 2017 only, indicating that highly diversified M. hyopneumoniae strains existed in Ontario. Some strains from the same geographic location but different years had the same sequence types, indicating that the same strain types circulate persistently in the field. Different p146 genotypes were also identified from similar geographic locations, indicating that either M. hyopneumoniae strains are prone to mutation or that multiple strains can infect the same or nearby swine production units.

Changes in antimicrobial susceptibility profiles of Mycoplasma bovis over time.

Mycoplasma bovis is a major cause of pneumonia, arthritis, and mastitis in cattle and can lead to significant economic losses. Antimicrobial resistance is a concern and further limits the already short list of drugs effective against mycoplasmas. The objective of this study was to examine changes in in vitro minimum inhibitory concentrations (MICs) of antimicrobials of aminoglycoside, fluoroquinolone, lincosamide, macrolide, pleuromutilin, phenicol, and tetracycline classes for 210 M. bovis isolates collected from 1978 to 2009. The MIC50 values of the various antimicrobials were also compared. The MIC50 levels for enrofloxacin and danofloxacin remained low (0.25 µg/mL) across all 3 decades. MIC50 levels for tetracyclines, tilmicosin, and tylosin tartrate were low in the 1980s, then increased in the 1990s and remained high. In the 1980s, MIC50 levels were low for clindamycin, spectinomycin, and tulathromycin, increased in the 1990s to 8 µg/mL (clindamycin) and 32 µg/mL (spectinomycin and tulathromycin), then decreased again in the 2000s. Members of the fluoroquinolone class of antimicrobials had the lowest MIC50 levels across all 3 decades, which suggests in vitro susceptibility of M. bovis to this class of antimicrobials. Statistically significant associations were observed between MIC values for chlortetracycline, oxytetracycline, tylosin tartrate, and tilmicosin; between clindamycin, tulathromycin, spectinomycin, and tiamulin; and between tylosin tartrate and clindamycin. Changes in MIC levels of various antimicrobials over time show the importance of monitoring the susceptibility of mycoplasmas to antimicrobials. The number of antimicrobials that showed elevated MIC50 levels, and therefore possibly reduced in vitro effectiveness against M. bovis, supports initiatives that promote prudent use of antimicrobials in agriculture.

Mycoplasma bovis est une cause majeure de pneumonie, d'arthrite, et mammite chez les bovins et peut entrainer des pertes économiques significatives. La résistance antimicrobienne est une préoccupation et réduit encore plus la courte liste déjà existante de médicaments efficaces contre les mycoplasmes. L'objectif de la présente étude était d'examiner in vitro les changements des concentrations minimales inhibitrices (CMI) des antimicrobiens des classes des aminoglycosides, des fluoroquinolones, des lincosamides, des macrolides, des pleuromutilines, des phénicoles, et des tétracyclines envers 210 isolats de M. bovis collectionnés entre 1978 et 2009. Les valeurs de CMI50 des différents antimicrobiens ont également été comparées. Les valeurs de CMI50 de l'enrofloxacine et de la danofloxacine sont demeurées faibles (0,25 µg/mL) au cours des trois décennies. Les valeurs de CMI50 pour les tétracyclines, le tilmicosin et le tartrate de tylosine étaient basses dans les années 1980s, puis augmentèrent dans les années 1990s et sont demeurées élevées. Durant les années 1980s, les valeurs de CMI50 étaient basses pour la clindamycine, la spectinomycine, et la tulathromycine, augmentèrent dans les années 1990s jusqu'à 8 µg/mL (clindamycine) et 32 µg/mL (spectinomycine et tulathromycine), puis ont diminué encore dans les années 2000s. Les membres de la classe des fluoroquinolones avaient les valeurs de CMI50 les plus faibles au cours des trois décennies examinées, ce qui suggère une sensibilité in vitro de M. bovis à cette classe d'antibiotiques. Des associations statistiquement significatives furent notées entre les valeurs de CMI de la chlortétracycline, l'oxytétracycline, le tartrate de tylosine, et le tilmicosin; entre la clindamycine, la tulathromycine, la spectinomycine, et la tiamulin; et entre le tartrate de tyloosine et la clindamycine. Les changements dans les valeurs de CMI de différents antibiotiques dans le temps démontrent l'importance de suivre la sensibilité des mycoplasmes aux antimicrobiens. Le nombre d'antimicrobiens qui a démontré des valeurs élevées de CMI50, et ainsi une efficacité in vitro réduite envers M. bovis, encourage les initiatives qui font la promotion de l'usage prudent des antimicrobiens en agriculture.(Traduit par Docteur Serge Messier).

HIGH PREVALENCE OF MYCOPLASMA AND EIMERIA SPECIES IN FREE-RANGING EASTERN WILD TURKEYS ( MELEAGRIS GALLOPAVO SILVESTRIS) IN ONTARIO, CANADA.

Following extirpation from Ontario, Canada in the early 1900s, Eastern Wild Turkeys (EWTs; Meleagris gallopavo silvestris) were successfully reintroduced to the province in 1984. Despite the subsequent establishment of robust populations and biannual hunting seasons, data on the circulation of potential pathogens in these birds are lacking. Similarly, the interface between EWTs and poultry is poorly understood and includes possible bidirectional pathogen transmission via direct or indirect contact. Mycoplasma and Eimeria spp. are potential pathogens in Galliformes, and our objective was to determine their prevalence and distribution in Ontario EWTs. During the 2015 spring hunting season (April and May), oropharyngeal swabs from 147 hunter-harvested and five opportunistically collected EWTs from southern Ontario were cultured for Mycoplasma spp. The intestinal or cloacal contents of 107 of these birds and an additional 24 opportunistically and biologist-collected EWTs were analyzed for Eimeria spp. using PCR or fecal flotation. At least one Mycoplasma spp. was isolated from 98.7% (150/152) of EWTs, with six species identified. Mycoplasma gallopavonis was identified most commonly in 96.7% (147/152), followed by Mycoplasma gallinaceum in 23.7% (36/152). Potential poultry pathogens ( Mycoplasma meleagridis, Mycoplasma iowae, and Mycoplasma synoviae) were isolated from swabs of five (3.3%) EWTs. Coinfections with up to three Mycoplasma spp. were detected in 36.8% (56/152) of EWTs. Most EWTs tested positive for Eimeria spp. oocysts (75.6%; 99/131). A subset of positive samples ( n=16) were characterized by PCR, which detected the following species: Eimeria meleagrimitis (93.8%), Eimeria adenoeides (93.8%), Eimeria gallopavonis (56.3%), and Eimeria meleagridis (12.5%). The majority (93.8%) of these samples were positive for more than one Eimeria spp. We showed that numerous, mostly nonpathogenic Mycoplasma and Eimeria spp. circulate in EWTs across southern Ontario, and this helped to establish baseline information for comparison with future surveillance and monitoring.

Enhanced resistance to bacterial infection in protegrin-1 transgenic mice.

Antibiotic-resistant bacteria have become a public health concern. It was suggested that one source of resistant pathogens may be food-producing animals. Alternative approaches are therefore needed to enhance the resistance of farm animals to bacterial infection. Protegrin-1 (PG-1) is a neutrophil-derived antimicrobial peptide that possesses activity against a wide range of bacteria and enveloped viruses. Here we report on the production of transgenic mice that ectopically expressed PG-1 and compare their susceptibilities to Actinobacillus suis infection with those of their wild-type (WT) littermates. Of the 126 mice that were challenged with A. suis, 87% of the transgenic mice survived, whereas 31% of their WT littermates survived. The PG-1 transgenic mice had significantly lower bacterial loads in their lungs and reduced numbers of pulmonary pathological lesions. The antimicrobial function of PG-1 was confirmed in vitro by using fibroblast cells isolated from the transgenic mice but not the WT mice. Moreover, differential blood cell counts in bronchoalveolar lavage fluid indicated greater number of neutrophils in PG-1 transgenic mice than in their WT littermates after bacterial challenge. Our data suggest that the ectopic expression of PG-1 in mice confers enhanced resistance to bacterial infection, laying the foundation for the development of livestock with improved resistance to infection.

Development and field validation of a Mycoplasma iowae real-time polymerase chain reaction assay.

A Mycoplasma iowae real-time polymerase chain reaction (PCR) assay using primers and probes targeting the 16S rRNA gene was developed and field-validated in this study. The assay specifically identified M. iowae with a detection limit of 80 colony-forming units (cfu) per turkey cloacal swab sample (3.2 cfu per PCR reaction). It was validated by testing 154 field turkey cloacal swab samples in parallel with culture isolation. The diagnostic sensitivity of the PCR was 97.6%, and the specificity was 95.5%. The real-time PCR developed in this study is a rapid, sensitive, and cost-effective alternative to culture isolation for detecting M. iowae from cloacal swab samples.

The emergence of a new strain of porcine circovirus-2 in Ontario and Quebec swine and its association with severe porcine circovirus associated disease--2004-2006.

In the late fall of 2004 more severe lesions of porcine circovirus-2 associated disease (PCVAD) than usual occurred during an outbreak of porcine circovirus-2 (PCV-2) infection in Ontario nursery and grower/finisher pigs. The lesions were of unprecedented severity and included diffuse bronchointerstitial pneumonia, granulomatous enteritis, vasculitis, interstitial nephritis, and new lesions of splenic infarction. Some affected herds had up to 50% mortality. The outbreak correlated with the sudden emergence of a variant PCV-2, with PCR restriction fragment length polymorphism (RFLP) type 321. Phylogenetic comparison of ORF2 sequences and full genome sequences showed the new variant to be different from the previously dominant RFLP type 422 viruses, and similar to viruses that had occurred in France and other European and Asian countries. A subsequent retrospective study showed a statistically significant increase in the frequency of histological lesions in lymph node, spleen, lung, small intestine, colon and kidney, for pigs spontaneously infected with RFLP type 321, compared with the older RFLP type 422 strain. Viral burden, based on IHC staining in lymph node, also showed a statistically significant increase in pigs infected with the newer variant RFLP type 321, compared with the older RFLP type 422 strain. This enhanced virulence in pigs infected with PCV-2 RFLP type 321 strain may be related to the genetic differences in this new strain of PCV-2. This virus is now the dominant strain of PCV-2 virus found in Ontario and Quebec swine.

Multi-locus sequence types of Mycoplasma bovis isolated from Ontario, Canada in the past three decades have a temporal distribution.

A total of 217 Mycoplasma bovis isolates cultured from clinical cases in Ontario, Canada, over the past 30 y were selected to be characterized by a multi-locus sequence typing (MLST) method. Eleven housekeeping genes were evaluated for suitability for MLST; 2 loci that had been used in prior MLST schemes, dnaN and metS, along with hsp70 were chosen for further sequence analysis. The remaining loci- adk, efp, gmk, gyrB, polC, rpoB, tpiA, and uvrC genes-were not used because they had little to no sequence variation. The sequence data from the chosen loci ( dnaN, hsp70, metS) generated 28 sequence types (STs), with the 3 loci having 15, 5, and 7 alleles, respectively. These molecular typing results revealed that the STs had a temporal distribution; over the course of 3 decades, some STs disappeared and new STs appeared. Recent isolates had a greater variety of STs, which may indicate that new strains are emerging more rapidly now than in the past.