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Arthritis and Osteomyelitis Associated with Enterococcus Cecorum Infection in Broiler and Broiler Breeder Chickens in Ontario, Canada

In August 2008, an Ontario broiler chicken flock experienced an outbreak of lameness in 4-week-old birds, with morbidity reaching 7% by day 3. Necropsy examination and histopathology revealed arthritis of the hock, stifle, and coxofemoral joints, and femoral and vertebral osteomyelitis. Enterococcus cecorum was isolated from the lesions and identified by 16S ribosomal RNA sequencing. In October 2008, a second case of E. cecorum osteomyelitis involved a flock of 9-week-old broiler breeder chickens, with 2% of the male birds showing reluctance to walk. Necropsy examination revealed osteomyelitis and abscessation of the body of the caudal thoracic vertebra in affected birds, with impingement on the overlying spinal cord.

The first description of a (1 → 6)-β-D-glucan in prokaryotes: (1 → 6)-β-D-glucan is a common component of Actinobacillus suis and is the basis for a serotyping system.

Abstract The chemical and antigenic properties of the cell-surface lipopolysaccharides (LPSs) and capsular polysaccharides (CPSs) of seven representative strains of Actinobacillus suis from healthy and diseased pigs were investigated. Four strains produced a linear (1→6)-β- d -glucan homopolymer, β- d -Glc p -(1-[→6)-β- d -Glc p -(1-] n →, as a LPS-O-chain (O1) and as a CPS (K1). Polyclonal antisera prepared against a (1→6)-β- d -glucan-containing strain showed a positive reaction against both LPSs and CPSs derived from the above strains (designated serotype O1/K1). One strain carried the (1→6)-β- d -glucan solely as a LPS-O-chain (serotype O1) and two strains did not express the (1→6)-β- d -glucan, but, instead, produced a different O-chain (designated serotype O2); these three strains expressed their own characteristic CPSs. (1→6)-β- d -Glucan structures are common cell wall components of yeast, fungi and lichens, but, to our knowledge, this is the first time a (1→6)-β- d -glucan has been described in a prokaryotic organism. Conformational and nuclear magnetic resonance analyses showed that the β- d -Glc p -(1→6)-β- d -Glc p linkage was flexible and two distinct glycosidic conformers are described. Cross-reactive antibodies to the A. suis (1→6)-β- d -glucan could be detected in sera from a variety of species and in sera from specific pathogen free pigs. This cross-reactivity may arise from immuno-stimulation of organisms present in the surrounding environment that contain (1→6)-β- d -glucan, which may also explain the high incidence of false positive results in previous serological tests for A. suis . In addition, these (1→6)-β- d -glucan background antibodies may be protective against A. suis infection. The characterization herein of (1→6)-β- d -glucan is the foundation for the development of a serotyping system for A. suis .

Diversity of Enterococcus cecorum from chickens.

Enterococcus cecorum is a normal inhabitant of the intestine of birds and other vertebrates. It has recently emerged in Canada and other countries as an important cause of arthritis and osteomyelitis in chickens. The objectives of this study were to assess if this emergence was caused by a particular clone of E. cecorum and to assess the antimicrobial susceptibility of this organism. One hundred and thirteen E. cecorum isolates from infections in Canadian chickens (cases) and from the ceca of control chickens from Canada and Belgium were examined. Isolates were identified using biochemical tests and, for a number of them, identification was confirmed by partial 16S rRNA gene sequencing. Case and control isolates were typed by pulsed-field gel electrophoresis and tested for antimicrobial susceptibility using the broth microdilution method. Cecal isolates from control birds were genetically very diverse but the vast majority of those from cases belonged to a single major clonal lineage. Reduced susceptibility was widespread for tetracycline, bacitracin, and erythromycin. Isolates from cases were generally less susceptible to antimicrobial agents than isolates from control birds.

CLONAL EXPANSION OF THE PSEUDOGYMNOASCUS DESTRUCTANS GENOTYPE IN NORTH AMERICA IS ACCOMPANIED BY SIGNIFICANT VARIATION IN PHENOTYPIC EXPRESSION

Pseudogymnoascus destructans is the causative agent of an emerging infectious disease that threatens populations of several North American bat species. The fungal disease was first observed in 2006 and has since caused the death of nearly six million bats. The disease, commonly known as white-nose syndrome, is characterized by a cutaneous infection with P. destructans causing erosions and ulcers in the skin of nose, ears and/or wings of bats. Previous studies based on sequences from eight loci have found that isolates of P. destructans from bats in the US all belong to one multilocus genotype. Using the same multilocus sequence typing method, we found that isolates from eastern and central Canada also had the same genotype as those from the US, consistent with the clonal expansion of P. destructans into Canada. However, our PCR fingerprinting revealed that among the 112 North American isolates we analyzed, three, all from Canada, showed minor genetic variation. Furthermore, we found significant variations among isolates in mycelial growth rate; the production of mycelial exudates; and pigment production and diffusion into agar media. These phenotypic differences were influenced by culture medium and incubation temperature, indicating significant variation in environmental condition - dependent phenotypic expression among isolates of the clonal P. destructans genotype in North America.

A prospective study of sheep and goat abortion using real-time polymerase chain reaction and cut point estimation shows Coxiella burnetii and Chlamydophila abortus infection concurrently with other major pathogens

From 2009 to 2011, 163 sheep and 96 goat abortion submissions were received at the Animal Health Laboratory, University of Guelph, Ontario, Canada, for gross and histologic examination, as well as real-time polymerase chain reaction (PCR) testing for Chlamydophila abortus and/or Coxiella burnetii. Additional testing included immunohistochemistry for Toxoplasma gondii and Chlamydophila spp., routine bacterial culture and selective culture for Campylobacter spp., examination of modified acid-fast–stained placenta smears, enzyme-linked immunosorbent assay testing for Chlamydophila spp., and virus isolation. The final diagnosis made for each case by individual pathologists, based on gross and histologic lesions, as well as ancillary testing, was used as a standard to determine the significance of C. abortus and C. burnetii infection. Coxiella burnetii was identified by real-time PCR in 113 of 163 (69.0%) and 72 of 96 (75%) sheep and goat abortion submissions, respectively, but was considered to be significant in causing abortion in only 11 of 113 (10%) sheep and 15 out of 72 (21%) goat submissions that tested positive. Chlamydophila abortus was identified by real-time PCR in 42 of 162 (26%) and 54 of 92 (59%) sheep and goat submissions, respectively, but was considered the cause of the abortion in 16 of 42 (38%) sheep and 34 of 54 (63%) goat submissions that tested positive. Optimal sensitivity and specificity cut points for the real-time PCR copy number for C. abortus and C. burnetii were determined using the final pathology diagnosis as the reference test.

Beta 2 Toxigenic Clostridium Perfringens Type A Colitis in a Three-Day-Old Foal

Beta 2 (β2)-toxigenic Clostridium perfringens type A was recovered in large numbers from the intestine of a neonatal foal with colitis. The foal had been treated with gentamicin. Necropsy revealed marked distension of cecum and colon with watery, rust-colored homogeneous fluid and gastric infarction. Microscopic colonic lesions were superficial necrosis of 50% of the colonic mucosal surface and scattered 1–3-mm ulcers with subjacent neutrophilic infiltration and large Gram-positive bacilli in the necrotic mucosa. Beta-2 toxin was demonstrated in the lesions by immunohistochemical staining.

Multiple-class antimicrobial resistance surveillance in swine Escherichia coli F4, Pasteurella multocida and Streptococcus suis isolates from Ontario and the impact of the 2004–2006 Porcine Circovirus type-2 Associated Disease outbreak

The objective of this work was to describe trends in multiple-class antimicrobial resistance present in clinical isolates of Escherichia coli F4, Pasteurella multocida and Streptococcus suis from Ontario swine 1998-2010. Temporal changes in multiple-class resistance varied by the pathogens examined; significant yearly changes were apparent for the E. coli and P. multocida data. Although not present in the E. coli data, significant increases in multiple-class resistance within P. multocida isolates occurred from 2003 to 2005, coinciding with the expected increase in antimicrobials used to treat clinical signs of Porcine Circovirus Associated Disease (PCVAD) before it was confirmed. Prospective temporal scan statistics for multiple-class resistance suggest that significant clusters of increased resistance may have been found in the spring of 2004; months before the identification of the PCVAD outbreak in the fall of 2004.

Complete Genome Sequences of 17 Canadian Isolates of Salmonella enterica subsp. enterica Serovar Heidelberg from Human, Animal, and Food Sources

ABSTRACT Salmonella enterica subsp. enterica serovar Heidelberg is a highly clonal serovar frequently associated with foodborne illness. To facilitate subtyping efforts, we report fully assembled genome sequences of 17 Canadian S. Heidelberg isolates including six pairs of epidemiologically related strains. The plasmid sequences of eight isolates contain several drug resistance genes.

Risk factors associated with the colonization of Ontario layer chicken flocks with Brachyspira species.

Brachyspira species are frequent colonizers of the gastrointestinal tract in a variety of domestic animals, including birds. In chickens, Brachyspira species are associated with a clinical condition known as avian intestinal spirochetosis (AIS), a disease characterized by chronic diarrhoea, weight loss, low egg production, and faecal-stained eggs. The purpose of this study was to identify risk factors associated with the presence of Brachyspira species in Ontario layer chicken flocks. Pooled faecal samples were collected from 89 flocks from 58 farms between August 2010 and February 2011; 52 flocks were classified as dirty flocks (history of downgrades for dirty eggs) and 37 were classified as clean flocks (no history of downgrades for dirty eggs). A questionnaire related to management, biosecurity practices, and antimicrobial use was administered prior to sample collection. Using real-time polymerase chain reaction; 63.5% of the dirty flocks and 24.3% of the clean flocks were positive for Brachyspira species. A logistic regression model with a random effect for farm showed that the odds of Brachyspira species for flocks ≥ 60 weeks of age were higher than for flocks ≤ 34 weeks (OR=9.3; P=0.014). The odds of Brachyspira species in flocks housed in A-frame cages with manure curtains (OR=20.0; P=0.002) and flocks from multi-age farms (OR=8.5; P=0.001) were higher than for flocks in cage-stacked houses and from single-age farms, respectively. The odds of Brachyspira species for flocks housed in barns ≥ 30 years old was lower than for flocks housed in barns ≤ 14 years old (OR=0.1; P=0.002). The calculated intra-class correlation coefficient was 5.6 × 10(-14); the notably low proportion of variation among farms after the fixed effects were included in the model suggests that the farm-level variable (multi-age farm) included in the final model accounted for most of the farm-to-farm variation in Brachyspira presence. Therefore, it is recommended that strict biosecurity, and between-flock decontamination efforts to reduce the infection pressure, be followed on farms with multiple flocks of different ages to avoid transmission of the bacteria between flocks.

Species level identification of coagulase negative Staphylococcus spp. from buffalo using matrix-assisted laser desorption ionization–time of flight mass spectrometry and cydB real-time quantitative PCR

Incorrect identification of Staphylococcus spp. can have serious clinical and zoonotic repercussions. Accordingly, the aim of this study was to determine if matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and/or cydB real- time quantitative PCR (qPCR) could be used to accurately identify coagulase negative Staphylococcus spp. (CoNS) obtained from buffalo milk and milking environment samples. Seventy-five of 84 CoNS isolates could be identified to the species level (score value >1.99) using MALDI-TOF MS. However, as determined by cytochrome d ubiquinol oxidase subunit II (cydB) qPCR and by 16S RNA and cydB gene sequencing, 10S. agnetis strains were wrongly identified as S. hyicus by MALDI-TOF MS. In addition, 9 isolates identified by MALDI-TOF only to the genus level (score values between 1.70 and 1.99) could be identified to species by cydB qPCR. Our findings suggest that MALDI-TOF MS is a reliable method for rapid identification of S. chromogenes and S. epidermidis (species of interest both in human and veterinary medicine) and may be able to correctly identify other Staphylococcus spp. However, at present not all Staphylococcus spp. found in buffalo milk can be accurately identified by MALDI-TOF MS and for these organisms, the cydB qPCR developed in the current study may provide a reliable alternative method for rapid identification of CoNS species.