CONTROL ID: 2021079

TITLE:Isolation of a Novel Campylobacter Species from Bovine Genital Sample

ABSTRACT BODY:

Narrative: Campylobacter fetus comprises two subspecies, C. fetus subsp. fetus and C. fetus subsp. venerealis. C. fetus subsp. venerealis causes bovine venereal campylobacteriosis (BVC), a sexually transmitted disease of cattle that if not properly controlled leads to severe economic loss particularly to industries with focus on breeding programs. Therefore, accurate identification of the organism is essential. C. fetus subsp. venerealis can be differentiated from closely related Campylobacter spp. by their growth behavior in 1% glycine and susceptibility pattern on cephalothin followed by confirmation with molecular method. At the Wisconsin Veterinary Diagnostic Laboratory we use a real-time polymerase chain reaction (rt-PCR) assay that simultaneously targets a gene that is present in C. fetus and a gene that is reportedly unique to C. fetus subsp. venerealis. Recently, a slow growing, gram-negative curved bacterium was recovered from bovine vaginal wash and swab. The organism exhibited C. fetus subsp. venerealis characteristics by biochemical and susceptibility patterns and was      negative by PCR for Campylobacter fetus but positive for C. fetus subsp. venerealis.

Comparative 16S sequence based identification analysis indicated that the isolate belongs to the genus Campylobacter. Cluster analysis derived from the 16S rDNA sequences revealed that the isolate is more closely related to other campylobacters than to C. fetus subsp. venerealis. Genotyping by Pulsed Field Gel electrophoresis (PFGE) did not produce meaningful results. Further, the matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) and cellular fatty acid methyl ester analysis yielded no identification. These findings suggest that the isolate may be novel and deserves further study as potential veterinary pathogen.

CURRENT CATEGORY/DISCIPLINE: Bacteriology/Mycology

AUTHORS/INSTITUTIONS: B. Angell, M. MacNab, O. Okwumabua, Veterinary Diagnostic Laboratory, University of Wisconsin, Madison, Wisconsin, UNITED STATES;

AWARDS:

Trainee Letter:

CONTROL ID: 2020773

TITLE:Isolation and Identification of Helcococcus ovis from Bovine Joint Fluid

ABSTRACT BODY:

Narrative: Lung tissue and joint fluid were submitted to the University of Missouri College  of Veterinary Medicine Veterinary Medical Diagnostic Laboratory for Bacteriology and Molecular Diagnostic examination. The samples were obtained from a Holstein calf with clinical signs of possible pneumonia and swollen joints. As per standard operational procedure the submitted samples were cultured aerobically on Blood and MacConkey agars as well as under microaerophilic conditions (5% CO2, 95% air) on Chocolate and Eugon Chocolate Agars. Both submitted samples were found to contain mixed microbial growth. The lung contained light growth of isolates subsequently identified as Corynebacterium,  Staphylococcus and Streptococcus species. The joint fluid contained light growth of a hemolytic Bacillus on aerobic culture and heavy growth of minute grey colonies on Chocolate and Eugon Chocolate agars cultured in the presence of CO2. This latter isolate was found to  be KOH and catalase negative and appeared microscopically as paired Gram-positive cocci resembling Micrococcus. Automated bacterial identification systems (Thermo Fisher Scientific Sensititre AP90 Gram-positive Identification system) failed to identify the isolate even           to the level of genus. Ribosomal RNA (16S) sequencing was employed as a diagnostic tool    to identify this unusual microbe. Results from sequencing showed one nucleotide       difference out of 1423 base pairs between this isolate and the Helcococcus ovis type strain s840-96-2. Helcococcus ovis, a facultative anaerobic Gram positive catalase negative cocci

was first recovered together with Trueperella pyogenes from multiple tissues during post mortem examination of two adult sheep in Scotland. Like that report we have demonstrated an aerobic growth dependency on Staphylococcus aureus and are unaware of the clinical significance of this isolate in the joint fluid of the bovine. Independent molecular evaluation of the submitted samples demonstrated that the lung was PCR positive for Mycoplasma, but not

M. bovis; the joint fluid was Mycoplasma negative by PCR. Microbiological and molecular testing is still ongoing.

CURRENT CATEGORY/DISCIPLINE: Bacteriology/Mycology

AUTHORS/INSTITUTIONS: W. Fales, J.W. Bowman, I.K. Ganjam, D. Kim, S. Schommer,

M.J. Calcutt, T.J. Reilly, Department of Veterinary Pathobiology, University of Missouri, Columbia, Missouri, UNITED STATES;

AWARDS:

Trainee Letter:

CONTROL ID: 2019343

TITLE: A Taqman Multiplex Real-Time PCR for Rapid Detection of Viral and Bacterial Pathogens Associated with Bovine Respiratory Disease Complex

ABSTRACT BODY:

Narrative: Bovine Respiratory Disease Complex (BRDC) is the most significant infectious disease in cattle. The disease is multifactorial, with either stress or reduced immunity allowing primary and/or secondary infections by viral and bacterial pathogens to occur. Bovine viral diarrhea virus (BVDV), Bovine respiratory syncytial virus (BRSV), Bovine coronavirus (BCoV) and Bovine herpesvirus type 1 (BHV-1/IBR), Mannheimia haemolytica (Mh), Pasteurella multocida (Pm), Histophilus somni (Hs), Bibersteinia trehalosi (Bt) and Mycoplasma bovis (Mb) are 9 pathogens that are commonly associated with BRDC. A Taqman multiplex real-time PCR assay consisting of three triplex reactions was developed and validated for simultaneous detection of these 9 pathogens involved in BRDC. The complete panel includes a triplex reaction for detection of the BRD viral RNA pathogens (BVD,    BRSV and BCoV), a triplex reaction for DNA pathogens (M. bovis, BHV1/IBR and Pasteurella multocida) and the third triplex assay for the detection of the BRD bacterial pathogens: M. haemolytica, H. somni and B. trehalosi. Each individual singleplex reaction  was optimized separately and subsequently multiplexed, with PCR amplification efficiencies of 92.1-105.4% and correlation coefficients of 0.99-0.999. Assay specificity was assessed by testing non-target and closely related species and there was no cross-reactivity observed.

Comparison of detection limit and reaction efficiency between singleplex and multiplex assays indicated that multiplexing does not inhibit the detection sensitivity of the assay.            Testing on 70 clinical samples using the assay showed good sensitivity and specificity. This study provides the basis for further evaluating the assay’s diagnostic performance in clinical service for rapid detection of viral and bacterial pathogens associated with Bovine Respiratory Disease Complex (BRDC).

CURRENT CATEGORY/DISCIPLINE: Bacteriology/Mycology | Virology

AUTHORS/INSTITUTIONS: Q. Sun, A. Rebelo, L. Peddireddi, R. Hesse, R.D. Oberst, B. Lubbers, G. Anderson, J. Bai, Veterinary Diagnostic Laboratory, Kansas State University, Manhattan, Kansas, UNITED STATES;

AWARDS:

Trainee Letter:

CONTROL ID: 2022871

TITLE: Development and Evaluation of a Real-Time PCR Assay for Detection of

Salmonella Heidelberg from Clinical and Environmental Samples

ABSTRACT BODY:

Narrative: Salmonella Heidelberg represents an emerging health concern both for veterinary patients and humans due to zoonotic transmission. Recent food-borne outbreaks related to this serotype have also demonstrated the potential hazard it presents to the safety of our human food supply. While there are currently no demands for regulatory testing related to S. Heidelberg, management and testing requirements may be forthcoming in an attempt to reduce its impact from livestock and poultry food product sources. Twenty-one S. Heidelberg isolates along with 21 non-S. Heidelberg isolates recovered from environmental and clinical submissions to the California Animal Health and Food Safety Lab System (CAHFS) were submitted for whole-genome sequencing and analysis was performed to determine if a  suitable gene target could be identified for PCR detection. After preliminary evaluation in silico, and a putative Type II restriction enzyme sequence was selected for further evaluation. A real-time PCR assay was developed that identified a 62 base-pair target in this region conserved in S. Heidelberg but not present in other serotypes. PCR testing on a panel of 26 different S. Heidelberg isolates demonstrated 100% detection utilizing this target. When tested against 22 non-Heidelberg Salmonella sp. organisms, 21 of the strains generated          negative results to this target. The single discrepant result yielded a PCR product from a S. Newport isolate with a Ct of 36.8 in which a large concentration of DNA (403 ng/µl) was tested; dilution of this sample to a concentration closer to that obtained for other isolates tested in this assay (140 ng/µl) did not result in a detectable PCR product on repeat testing.

Consistent detection was obtained with concentrations of 1.5 X 103 cfu/ml in broth samples without enrichment. No interference was seen when samples were co-inoculated with S. Heidleberg and non-Heidelberg Salmonella serotypes. Side by side comparisons between this real-time PCR assay and traditional culture with serotyping performed on tetrathionate broths from twenty field samples incubated for 18-24 hours at 42 °C yielded 100% agreement between the two methods. This assay may prove to be useful for sensitive and specific detection of S. Heidelberg in clinical and environmental samples.

CURRENT CATEGORY/DISCIPLINE: Bacteriology/Mycology | Epidemiology

AUTHORS/INSTITUTIONS: K. Clothier, A. Torain, S. Reinl, California Animal Health and Food Safety Laboratory, University of California, Davis, California, UNITED STATES; K. Clothier, Department of Pathology, Microbiology, and Immunology, School of Veterinary Medicine, University of California, Davis, California, UNITED STATES;

B. Weimer, Department of Population Health and Reproduction, School of Veterinary Medicine, University of California, Davis, California, UNITED STATES;

AWARDS:

Trainee Letter:

CONTROL ID: 2022550

TITLE: Prevalence, isolation and characterization of E. coli O104 in cattle feces

ABSTRACT BODY:

Narrative: The hybrid serotype of E. coli O104:H4, reported in 2011 outbreak in Germany, possessed characteristics of two pathotypes, Shiga toxin-producing and enteroaggregative E. coli. Although O104:H4 has not been detected in cattle, other serotypes (O104:H21, O104:H2, O104:H7, O104:H12) have been detected in cattle and other animals. The objectives of our study were to determine the prevalence of E. coli O104 in feedlot cattle feces, isolate and characterize the strains. A total of 757 rectal content samples from cattle, representing 29 different feedyards, were collected at a slaughter plant in July, 2013. Fecal samples were enriched in E. coli broth for 6 h at 40 C and then subjected to PCR- and culture-based methods

of detection. DNA extracted from pre and post enriched fecal samples were tested by a multiplex PCR to detect serogroup O104 and associated virulence genes (terD, ehxA, stx1, stx2, bfpA, aggA, eae and flicH4) of the hybrid pathotype. Culture-based method of detection involved immunomagnetic separation with O104 beads, plating on selective chromogenic medium, followed by serogroup confirmation by PCR. Isolates of O104 were tested by PCR assays to determine capsular antigen of O8/O9 serogroups, virulence genes and flagellar types. Of the 757 samples, 38(5%) and 349 (46%) samples were positive before and after enrichment respectively for O104 serogroup-specific gene. A total of 143 O104 isolates were obtained and 92 of them were positive for O8/O9 capsular antigen genes. Of the 51 O104 isolates, only 16 (31.4%) carried stx1, none of them carried eae. Subtyping of stx1 was performed based on amino acid sequences and all 16 isolates carried stx1c. In silico restriction fragment length polymorphism (RFLP) was performed to compare with the subtyping based on amino acid sequences. In silico RFLP was also compared with conventional PCR-RFLP. The results from PCR/RFLP and in silico RFLP were in agreement with those obtained by subtyping based on amino acid sequences. The O104 isolates harbored diverse flagellar (H) antigens with 36 isolates containing H7, 4 H2, 1 each of H21 and H1. Pulsed-field gel electrophoresis (PFGE) was performed to assess the genetic relatedness of the stx-positive O104 isolates. The 16 stx1- positive isolates formed three PFGE clusters and the 13 isolates from one feedyard were of the same PFGE subtype (100% similarity). Cattle shed serogroup O104 in feces, but only a few strains (11.2 %) carried stx1 gene and none of the isolated strains carried genes characteristic  of the hybrid pathotype.

CURRENT CATEGORY/DISCIPLINE: Epidemiology

AUTHORS/INSTITUTIONS: P. B. Shridhar, L.W. Noll, X. Shi, N. Cernicchiaro, J. Bai,

D.G. Renter, T. Nagaraja, Department of Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, Kansas, UNITED STATES;  P. B. Shridhar, J. Bai, Vaterinary Diagnostic Laboratory, College of Veterinary Medicine, Kansas State university, Manhattan, Kansas, UNITED STATES;

AWARDS: Graduate Student Oral Presentation

Trainee Letter: trainee letter-2014.pdf

CONTROL ID: 2021612

TITLE:An Improved Primer Set for the Genotyping of Clostridium perfringens

ABSTRACT BODY:

Narrative: Clostridium perfringens is a widely distributed Gram-positive bacterium responsible for a wide range of diseases in humans and animals. C. perfringens strains are categorized into five toxinotypes (A-E) according to their complement of four major toxin genes. There is an important variation within the cpa gene, which encodes the alpha toxin present in all toxinotypes of C. perfringens. In addition, the gene encoding Beta2 toxin is present as two alleles with ~70% sequence homology and substantial sequence variation in each allele. Collectively, this presents a diagnostic challenge for accurately genotyping isolates for the cpa and cpb2 genes. This study focused on strain variation between isolates             from calves and the effect of this variation on genotyping accuracy. New primer sets for amplifying cpa and cpb2 were designed based on all known sequence variants of the cpa and cpb2 genes. The new primer set identified isolates carrying cpb2 and cpa in samples that did not show bands corresponding to these genes using previously published and widely used primer sets. This primer set represents an important improvement in the diagnostic genotyping of C. perfringens field isolates.

CURRENT CATEGORY/DISCIPLINE: Bacteriology/Mycology | Pathology

AUTHORS/INSTITUTIONS: B.N. Saeed, K. Mills, D. O'Toole, B. Schumaker, W. Laegreid, Department of Veterinary Sciences, University of Wyoming, Laramie, Wyoming,

UNITED STATES;

B. Bisha, Department of Veterinary Sciences, University of Wyoming, Laramie, Wyoming, UNITED STATES;

AWARDS: Graduate Student Poster Presentation|Graduate Student Oral Presentation

Trainee Letter:

CONTROL ID: 2021335

TITLE:Survey of Respiratory Pathology in Wild Urban Rats (Rattus norvegicus and Rattus rattus)

ABSTRACT BODY:

Narrative: Norway (Rattus norvegicus) and black rats (R. rattus) are common commensal pests that live in association with human habitats and carry a number of zoonotic pathogens, yet we know little about wild rat ecology, including their natural diseases. We describe the gross and histological lesions of the respiratory tract in a sample of 711 wild urban rats. Over a 1-year period, rats were trapped from an inner city neighborhood in Vancouver, Canada and autopsied. A subset was examined for 19 distinct categories of histological lesions in the respiratory tract. Samples were tested, using serology and PCR, for known rat respiratory pathogens. Grossly-evident lesions were rare (8/711; 1%). Upper respiratory tract (URT) inflammation (rhinitis, submucosal and periglandular lymphoplasmacytic tracheitis, and/or tracheal intraluminal necrotic debris) was present in 93/107 (87%) rats and was associated with Cilia Associated Respiratory Bacillus (CARB) and Mycoplasma pulmonis (P ≤ .05), and heavier rats (OR = 1.09, 95% CI = 1.05 – 1.14 per 10 g). Lower respiratory tract (LRT) inflammation (peribronchiolar and/or perivascular lymphoplasmacytic cuffing) was present in 152/199 rats (76%) and inflammation was associated with CARB and M. pulmonis (P ≤ .02), and heavier rats (OR = 1.20, 95% CI = 1.14 – 1.27 per 10 g). Pneumocystis carinii was detected in 48/102 (47%) rats using PCR, but was not significantly associated with lesions.

This is the first detailed description of pathology in the respiratory system of wild rats and demonstrates that respiratory disease is common. Although the impact of these lesions on individual and population health remains to be investigated, respiratory disease may be an important contributor to wild rat morbidity and mortality.

CURRENT CATEGORY/DISCIPLINE: Pathology | Bacteriology/Mycology

AUTHORS/INSTITUTIONS: J.L. Rothenburger, F.A. Leighton, Department of Veterinary Pathology, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, CANADA;

C.G. Himsworth, Animal Health Centre, British Columbia Ministry of Agriculture, Abbotsford, British Columbia, CANADA;

C.G. Himsworth, School of Population and Public Health, University of British Columbia, Vancouver, British Columbia, CANADA;

C.B. Clifford, Charles River, Wilmington, Massachusetts, UNITED STATES;

P.M. Treuting, School of Medicine, University of Washington, Seattle, Washington, UNITED STATES;

F.A. Leighton, National Headquarters, Canadian Wildlife Health Cooperative, Saskatoon, Saskatchewan, CANADA;

AWARDS: Trainee Travel Award|Graduate Student Poster Presentation|Graduate Student Oral Presentation

Trainee Letter: AAVLD Trainee Travel Awards Applicant Letter_JR.pdf

CONTROL ID: 2022690

TITLE: Minimal Inhibitory Concentration Antimicrobial Susceptibilities of Coagulase Positive Staphylococcus sp. Associated with Canine Pyoderma (2011-2013)

ABSTRACT BODY:

Narrative: Skin swabs or skin biopsies from dogs with pyoderma were submitted to the University of Illinois Veterinary Diagnostic Laboratory for bacterial culture and susceptibility testing as part of a study investigating potential for exchange of antimicrobial resistance and/or virulence factors between Staphylococcus species during mixed infections. Dominant among the isolates recovered were coagulase positive Staphylococcus species, predominantly Staphylococcus pseudintermedius (N=327), Staphylococcus schleiferi (N=38) and Staphylococcus aureus (N=17). Isolates were identified by Gram stain, catalase, coagulase, ONPG broth, and/or Sensititre™ GPID or Biolog™ GP2 systems as needed. Only 12 dogs had significant co-infections with multiple Staphylococcus species, S. pseudintermedius with

either S. aureus or S.schleiferi. Susceptibilities were performed using Sensititre™ Companion Animal 1F or 2F MIC panels, following Clinical Laboratory Standards Institute M31-A3 and Vet01-A4/S2 guidelines and interpretations. Penicillin resistance was common among S. pseudintermedius isolates (MIC50=1.0 μg/ml, MIC90=16.0 μg/ml) while S. schleiferi were more susceptible (MIC50≤ 0.06 μg/ml, MIC90=1.0 μg/ml). A similar trend was observed for ampicillin with a S. schleiferi MIC90 = 1.0 μg/ml and MIC90 ≥ 4.0 μg/ml for both S. pseudintermedius and S. schleiferi. Based upon oxacillin breakpoints of 4.0 μg/ml for S. aureus and 0.5 μg/ml for S. pseudintermedius and S. schleiferi, 35.3%, 30.4% and 34.2% of these isolates exhibited methicillin resistance, respectively. While the MIC50 values were all

0.25 μg/ml, MIC90 values were 8.0, 4.0 and 2.0, respectively. There was good correlation between the oxacillin breakpoints and the presence of the mecA gene detected by PCR. Fluoroquinolone resistance was more common among S. schleiferi isolates (MIC50=1.0 μg/ml

, MIC90=2.0 μg/ml ), and both S. aureus and S. pseudintermedius had MIC50 values of ≤0.25 μg/ml but MIC90 values of ≥ 4.0 μg/ml for both enrofloxacin and marbofloxacin. The prevalence of methicillin resistance among coagulase positive Staphylococci is much higher for dogs with pyoderma (~30%) than the prevalence reported for healthy dogs (~6% for methicillin resistance) possibly as a result of selection pressure associated with antibiotic treatment.

CURRENT CATEGORY/DISCIPLINE: Bacteriology/Mycology

AUTHORS/INSTITUTIONS: C.W. Maddox, Department of Pathobiology, University of Illinois, Urbana, Illinois, UNITED STATES;

K.L. Ellis, A.K. Stevenson, I. Sweeney, S. Lanka, C.W. Maddox, Veterinary Diagnostic Laboratory, University of Illinois, Urbana, Illinois, UNITED STATES;

AWARDS:

Trainee Letter:

CONTROL ID: 2022625

TITLE: Virulence Factors Genes (siet, lukI and lukF) in Staphylococcus Species Isolated from Canine Clinical Specimens

ABSTRACT BODY:

Narrative: The epidemiology and study of virulence factors in methicillin-resistance (MR) S. pseudintermedius and S. schleiferi subsp. coagulans isolated from dogs is sparse. The aim of this study was to investigated the presence of the genes siet, lukI and lukF, which encode for important virulence factors in S. schleiferi subsp. coagulans, S. pseudintermedius and S. aureus isolated from canine clinical specimens. In addition, related epidemiological information related with previous antimicrobial therapy, breed, gender, age and prevalence of methicillin-resistant isolates by year was analyzed. A total of 577 coagulase positive Staphylococcus species were collected from canine clinical specimens, and epidemiological information was collected from the clinical history submitted to the lab. The incidence of the genes siet, lukI and lukF were determined by PCR. A significant difference (p ≤ 0.05) indicated that isolates from dogs with a history of antimicrobial therapy have a higher risk of being MR positive, than from dogs without therapy. Within the S. aureus group, the panton-

valentine leukocidin genes were tested in the MRSA group with four testing positive, 12.9% (4/31). LUK-I and siet genes were found mainly in S. pseudintermedius, but also in S. aureus and S. schleiferi subsp. coagulans. An important finding in this study was the discovery of the genes siet and lukI in S. aureus and S. schleiferi subsp.coagulans. Collectively, these findings support the need for further epidemiological and clinical studies concerning virulence factors in Staphylococcus species of canine origin, which may be a potential hazard for public health.

CURRENT CATEGORY/DISCIPLINE: Bacteriology/Mycology | Epidemiology

AUTHORS/INSTITUTIONS: D.V. Diaz-Campos, Department of Veterinary Microbiology and Pathology, Animal Disease Diagnostic Laboratory, Washington State University, Pullman, Washington, UNITED STATES;

T. Hathcock, K.V. Brock, Department of Pathobiology, Auburn University, Auburn, Alabama, UNITED STATES;

R.A. Palomares, Department of Population Health, University of Georgia, Athens , Georgia, UNITED STATES;

AWARDS:

Trainee Letter:

CONTROL ID: 2010678

TITLE:Fatalities Caused By Streptococcus dysgalactiae subsp. equisimilis in Horses

ABSTRACT BODY:

Narrative: Streptococcus dysgalactiae subsp. equisimilis (S. equisimilis) causes severe clinical syndromes in humans with high mortality rates including arthritis, pleuropulmonary infections, peritonitis, intra-abdominal and epidural abscesses, meningitis, endocarditis,                puerperal septicemia, neonatal infections, necrotizing fasciitis, myositis, and streptococcal toxic-like syndrome. S. equisimilis has been reported in horses from aborted placenta, less frequently from abscessed lymph nodes, and the upper respiratory system infections in horses. However, fatalities caused by S. equisimilis in horses are poorly described and, comprehensive and exhaustive studies are limited. The goal of this study is to describe fatalities of horses caused by S. equisimilis and examine the genotypes of equine isolates of S. equisimilis by using multilocus sequence analysis, and M virulence protein gene (emm) typing combined  with phenotypic characterization.

105 equine necropsies where S. equisimilis were isolated were retrospectively analyzed. The necropsies were performed at the University of Kentucky Veterinary Diagnostic Laboratory between January 1, 2010 and December 31, 2013. In 22 cases, S. equisimilis was reported as only causative microorganism leading to death. In 15 of these cases, S. equisimilis caused abortion due to placentitis with or without fetal septicemia. S. equisimilis also caused osteoarthritis/ osteomyelitis/arthritis (5 cases), and septicemia (2 cases). In 29 other cases, S. equisimilis was diagnosed as contributing or associated agent to the fatality (or euthanasia) causing placentitis/abortion (9 cases), pleuritis (4 cases), pneumonia (4 cases), septicemia (4 cases), dermatitis/ cellulitis/ myelitis (3 cases), osteomyelitis/arthritis (2 cases), cystitis (1 case), omphalitis (1 case), and mesenteric abscess (1 case). Other contributing microorganisms identified in these cases were S. aureus, S.equi subsp. zooepidemicus, P. caballi, Neorickettsia risticii, R. equi, A. equi subsp. equi, K. pneumonia, E. coli, Enterococcus spp and Streptococcus spp. In 54 necropsy cases, S. equisimilis was recovered from tissues but not associated with any disease/fatality.

12 S. equisimilis isolates representing these groups were confirmed to be S. equisimilis by PCR using streptokinase gene specific primers. In addition, multilocus sequence types (MLSTs) and sequence types of the emm gene were obtained and compared to human disease case S. equisimilis isolates. These preliminary results shown that these S. equisimilis isolates recovered from equine necropsy cases have new MLSTs and emm types suggestive of a genetic lineage distinct from human isolate clonal complexes described to date.

CURRENT CATEGORY/DISCIPLINE: Bacteriology/Mycology

AUTHORS/INSTITUTIONS: E. Erol, L. Cassone, S. Locke, C. Jackson, C. Carter, Veterinary Diagnostic Laboratory, University of Kentucky, Lexington, Kentucky, UNITED STATES;

R.E. Gertz , B. Beall, Streptococcus Laboratory, CDC/NCIRD/DBD/RDB, Atlanta, Georgia, UNITED STATES;

O. Genc, Ondokuz Mayis University, Samsun, TURKEY;

AWARDS:

CONTROL ID: 2010731

TITLE:A Novel Avibacterium sp. Causes Mortality in Laying Hens

ABSTRACT BODY:

Narrative: In January 2014, a disease outbreak characterized by elevated mortality and reduced egg production occurred in a commercial in-line high-rise laying hen operation. White leghorn chickens in 3 of 9 houses on this farm were affected. Each house had an average capacity          of 111,000 birds and affected flocks ranged in age from 39 to 48 weeks. The flock          manager reported mildly swollen heads and conjunctivitis in a few hens housed in each of these three flocks. Macroscopic lesions consisted primarily of hepatosplenomegaly, air sacculitis, and pneumonia. Abundant caseous exudate was present in anterior thoracic air sacs and lungs were consolidated with exudate in bronchi. Livers, lungs, air sacs, and spleens were collected using aseptic technique and pooled. All samples were cultured using routine procedures on blood agar plates under aerobic and anaerobic conditions. Avibacterium sp. was isolated from lungs and   air sacs and E. coli was isolated from livers. Biochemical tests suggested that Avibacterium isolated from these chickens was not Avibacterium paragallinarum, the etiologic agent associated with infectious coryza. To identify the species of Avibacterium, the 16S ribosomal gene of selected isolates was amplified by PCR and sequenced. The 16S rRNA phylogenetic tree of veterinary Pasteurellaceae species confirmed that this isolate is not A. paragallinarum and is different from other Avibacterium sp. previously isolated from chickens in the United States.

CURRENT CATEGORY/DISCIPLINE: Bacteriology/Mycology

AUTHORS/INSTITUTIONS: D. Trampel, T. Frana, Department of Veterinary Diagnostic and Production Animal Medicine, Iowa State University, Ames, Iowa, UNITED STATES;

S.E. Tilley, M.E. Lee, Department of Population Health, University of Georgia, Athens, Georgia, UNITED STATES;

AWARDS:

Trainee Letter:

CONTROL ID: 2021346

TITLE: Development of a Multiplex PCR Assay for the Identification and Speciation of Pathogenic Vibrionaceae

ABSTRACT BODY:

Narrative: There are currently several emerging pathogens within the family Vibrionaceae that are causative agents of disease in both humans and fish. Identification and speciation of these organisms using classical culture and biochemical methods yield results that are frequently inconsistent, necessitating diagnostic techniques that are accurate, rapid, and cost effective for proper Vibrionaceae identification. Multiplex PCR has proven useful for Vibrio spp. in previous studies, although these tests have typically been limited to human pathogens such as Vibrio cholerae. We have designed a multiplex PCR assay for the detection of six fish and human pathogens within the Vibrionaceae family: Vibrio alginolyticus, Vibrio (Listonella)

anguillarum, Vibrio ordalii, Vibrio parahaemolyticus, Vibrio vulnificus, and Photobacterium damselae subsp. damselae. Each of these species was correctly and consistently identified when tested against their corresponding American Type Culture Collection (ATCC) strains and known clinical isolates, and no reaction was observed when tested against a variety of non-Vibrioisolates. Fifty-five presumptive Vibrio spp. clinical isolates were additionally tested by multiplex PCR, MALDI-TOF mass spectrometry, and sequencing of multiple gene targets. There was a high degree of concordance between mass spectrometry and multiplex            PCR results, though PCR showed a greater ability to differentiate between V. anguillarum and

V. ordalii isolates. The PCR assay demonstrated high accuracy compared to other available techniques and is also highly modular, as it can be successfully run as triplexes or a single hexaplex reaction and retains the potential of having additional primer sets incorporated into it for the detection of other Vibrio spp. This multiplex PCR assay represents a useful diagnostic tool for the proper identification of multiple Vibrionaceae species that will be beneficial for aquaculture and in both veterinary and human medicine.

CURRENT CATEGORY/DISCIPLINE: Bacteriology/Mycology

AUTHORS/INSTITUTIONS: S.D. Nydam, Paul G. Allen School for Global Animal Health, Washington State University, Pullman, Washington, UNITED STATES;

S.D. Nydam, C. Miller, T. Besser, K. Snekvik, Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, Washington, UNITED STATES;

C. Miller, T. Maddox, T. Besser, K. Snekvik, Washington Animal Disease Diagnostic Laboratory, Washington State University, Pullman, Washington, UNITED STATES;

K. Hammac, Indiana Animal Disease Diagnostic Laboratory, Purdue University, West Lafayette, Indiana, UNITED STATES;

AWARDS:

Trainee Letter: Seth Nydam Trainee Role.pdf

CONTROL ID: 2021359

TITLE: Use of Matrix-Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS) for the Speciation of Pathogenic Vibrioin Fish

ABSTRACT BODY:

Narrative: Many Vibrio spp. cause disease resulting in significant morbidity and mortality in farmed fish and significant economic losses to fish producers around the world. Speciation of Vibrio isolates is time consuming, difficult, and costly due to species diversity and inconsistency of phenotypic and biochemical traits within species. Therefore, improved methods of Vibrio speciation would aid identification accuracy and improve disease diagnosis. MALDI-TOF MS has been shown to be a rapid and cost effective method for speciation of numerous bacteria, but its accuracy for Vibriospp. identification has not been reported. Fifty- five presumptive Vibrio spp. isolates were subjected to three diagnostic approaches: MALDI- TOF MS (Bruker Biosciences Corp.), conventional aerobic bacteriology with biochemical phenotypic testing, and DNA sequence analyses using three conserved genes, rpoB, rpoD and toxR. DNA sequencing was presumed to be the most accurate and speciation by this method was completed on all 55 isolates. There was moderate agreement between the sequencing data and MALDI-TOF MS; however, certain species such as Vibrio ordalii, were inaccurately identified by MALDI-TOF MS. Thirty-four isolates (61.8%) had MALDI-TOF MS scores

>2.0, suggestive of accurate speciation and these included American Type Culture Collection (ATCC) strains of Listonella (Vibrio) anguillarum, Vibrio alginolyticus, Vibrio vulnificus, Vibrio ordalii, and Photobacterium damselae subsp. damselae. An additional five isolates (9.1%) were identified to the genus level with scores between 1.8 and 2.0. Two isolates (3.6%) produced scores <1.8 indicating inability to confidently assign genus or species. Fourteen isolates (25%) produced no score and thus no identification. It was also noted that the tube extraction method produced more consistent and higher quality scores compared to the direct transfer method. Conventional aerobic bacteriologic methods were applied to the same isolate