Enhancing the one health initiative by using whole genome sequencing to monitor antimicrobial resistance of animal pathogens: Vet-LIRN collaborative project with veterinary diagnostic laboratories in United States and Canada.

BACKGROUND: Antimicrobial resistance (AMR) of bacterial pathogens is an emerging public health threat. This threat extends to pets as it also compromises our ability to treat their infections. Surveillance programs in the United States have traditionally focused on collecting data from food animals, foods, and people. The Veterinary Laboratory Investigation and Response Network (Vet-LIRN), a national network of 45 veterinary diagnostic laboratories, tested the antimicrobial susceptibility of clinically relevant bacterial isolates from animals, with companion animal species represented for the first time in a monitoring program. During 2017, we systematically collected and tested 1968 isolates. To identify genetic determinants associated with AMR and the potential genetic relatedness of animal and human strains, whole genome sequencing (WGS) was performed on 192 isolates: 69 Salmonella enterica (all animal sources), 63 Escherichia coli (dogs), and 60 Staphylococcus pseudintermedius (dogs). RESULTS: We found that most Salmonella isolates (46/69, 67%) had no known resistance genes. Several isolates from both food and companion animals, however, showed genetic relatedness to isolates from humans. For pathogenic E. coli, no resistance genes were identified in 60% (38/63) of the isolates. Diverse resistance patterns were observed, and one of the isolates had predicted resistance to fluoroquinolones and cephalosporins, important antibiotics in human and veterinary medicine. For S. pseudintermedius, we observed a bimodal distribution of resistance genes, with some isolates having a diverse array of resistance mechanisms, including the mecA gene (19/60, 32%). CONCLUSION: The findings from this study highlight the critical importance of veterinary diagnostic laboratory data as part of any national antimicrobial resistance surveillance program. The finding of some highly resistant bacteria from companion animals, and the observation of isolates related to those isolated from humans demonstrates the public health significance of incorporating companion animal data into surveillance systems. Vet-LIRN will continue to build the infrastructure to collect the data necessary to perform surveillance of resistant bacteria as part of fulfilling its mission to advance human and animal health. A One Health approach to AMR surveillance programs is crucial and must include data from humans, animals, and environmental sources to be effective.

External ear cytological results and resident flora of clinically normal alpacas (Vicugna pacos).

BACKGROUND: Otitis is common in alpacas. Suppurative otitis media/interna can be an extension from the external ear canal or from a respiratory infection. Cytological evaluation provides rapid and inexpensive information to assist in therapeutic decision; to date, there is no published information regarding the normal cytological results and flora of the alpaca external ear canal. HYPOTHESIS/OBJECTIVES: To describe normal resident cytological findings and flora and possible variation over time, we sampled clinically normal alpaca external ear canals during two different seasons. ANIMALS: Fifty privately owned, healthy alpacas of different ages and sexes in two northeastern United States flocks. METHODS AND MATERIALS: One ear per alpaca had both cytological swabs (ectoparasites, inflammatory and epithelial cells, bacteria and yeast) and sterile swabs (bacterial and fungal cultures) taken. This was done in August 2017 and repeated in January 2018. RESULTS: Yeast organisms were noted cytologically in 2-4% of the samples. Prevalence of total yeast genera was 6% in August and 30% in January. Cytologically, rod-shaped bacteria [maximum 4-10/high power field (HPF); median 0-0.5/HPF] were seen in 50% of alpacas in August and 26% in January. Coccal bacteria (maximum 6-10/HPF; median 0/HPF) were seen in 32% of alpacas in August and 16% in January. No statistically significant findings were noted between sampling months. Common bacterial genera isolated in August were Bacillus (44%), Arthrobacter (40%) and nonhaemolytic Staphylococcus (26%), and in January were Bacillus (42%) and Pantoea (38%). CONCLUSIONS AND CLINICAL IMPORTANCE: This information may be useful when evaluating alpaca external ear canal samples, which subsequently may help dictate empirical therapy.

Comparative Phenotypic and Genotypic Analysis of Edwardsiella Isolates from Different Hosts and Geographic Origins, with Emphasis on Isolates Formerly Classified as E. tarda, and Evaluation of Diagnostic Methods.

Edwardsiella spp. are responsible for significant losses in important wild and cultured fish species worldwide. Recent phylogenomic investigations have determined that bacteria historically classified as Edwardsiella tarda actually represent three genetically distinct yet phenotypically ambiguous taxa with various degrees of pathogenicity in different hosts. Previous recognition of these taxa was hampered by the lack of a distinguishing phenotypic character. Commercial test panel configurations are relatively constant over time, and as new species are defined, appropriate discriminatory tests may not be present in current test panel arrangements. While phenobiochemical tests fail to discriminate between these taxa, data presented here revealed discriminatory peaks for each Edwardsiella species using matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) methodology, suggesting that MALDI-TOF can offer rapid, reliable identification in line with current systematic classifications. Furthermore, a multiplex PCR assay was validated for rapid molecular differentiation of the Edwardsiella spp. affecting fish. Moreover, the limitations of relying on partial 16S rRNA for discrimination of Edwardsiella spp. and advantages of employing alternative single-copy genes gyrB and sodB for molecular identification and classification of Edwardsiella were demonstrated. Last, sodB sequencing confirmed that isolates previously defined as typical motile fish-pathogenic E. tarda are synonymous with Edwardsiella piscicida, while atypical nonmotile fish-pathogenic E. tarda isolates are equivalent to Edwardsiella anguillarum Fish-nonpathogenic E. tarda isolates are consistent with E. tarda as it is currently defined. These analyses help deconvolute the scientific literature regarding these organisms and provide baseline information to better facilitate proper taxonomic assignment and minimize erroneous identifications of Edwardsiella isolates in clinical and research settings.

Lactococcus petauri sp. nov., isolated from an abscess of a sugar glider.

A strain of lactic acid bacteria, designated 159469T, isolated from a facial abscess in a sugar glider, was characterized genetically and phenotypically. Cells of the strain were Gram-stain-positive, coccoid and catalase-negative. Morphological, physiological and phylogenetic data indicated that the isolate belongs to the genus Lactococcus. Strain 159469T was closely related to Lactococcus garvieae ATCC 43921T, showing 95.86 and 98.08 % sequence similarity in 16S rRNA gene and rpoB gene sequences, respectively. Furthermore, a pairwise average nucleotide identity blast (ANIb) value of 93.54 % and in silico DNA-DNA hybridization value of 50.7  % were determined for the genome of strain 159469T, when compared with the genome of the type strain of Lactococcus garvieae. Based on the data presented here, the isolate represents a novel species of the genus Lactococcus, for which the name Lactococcus petauri sp. nov. is proposed. The type strain is 159469T (=LMG 30040T=DSM 104842T).

Effects of dexamethasone immunosuppression on turkey clostridial dermatitis.

Clostridia represents a group of anaerobic spore-forming bacteria ubiquitous in the poultry environment. They are widely distributed in soil and survive for many years as highly resistant, inactive spores. They enter the body through wounds and contaminated feed as active bacteria or spores. Multiplication of clostridial bacteria occurs only in the absence of oxygen or in environments with very low concentrations of oxygen. During active multiplication, the clostridial organisms produce several toxins that are responsible for most of the clinical signs seen in clostridial diseases. Immunosuppression is a problem for the poultry industry. In modern, intensive poultry-rearing conditions, stress due to high population densities pose a considerable challenge for the immune system, and infectious agents can exploit this situation to cause disease. Immunosuppression may predispose turkeys to clostridial infection, resulting in clostridial dermatitis and mortality. The purpose of this study was to determine whether immunosuppression predisposes turkeys to clostridial infection and causes clostridial dermatitis. We immunosuppressed 10-wk-old turkey poults with dexamethasone. The birds immunosuppressed and not immunosuppressed were then challenged with Clostridium perfringens, Clostridium septicum, or both and examined for the development of clostridial dermatitis. The dexamethasone-treated birds were found to be more susceptible to C. peifingens/C. septicum challenge and developed clostridial dermatitis than the no-dexamethasone-treated birds through the subcutaneous route. However, oral inoculation of the same agents did not cause any dermatitis lesions in either of the groups.

Characterization of immune responses and immunopathology in turkeys experimentally infected with clostridial dermatitis-producing strains of Clostridium septicum.

Clostridium septicum is one of the major causative agents of clostridial dermatitis (CD), an emerging disease of turkeys, characterized by sudden deaths and necrotic dermatitis. Despite its economic burden on the poultry industry, the immunopathological changes and pathogen-specific immune responses are poorly characterized. Here, we used three strains of C. septicum, namely Str. A1, Str. B1 and Str. C1, isolated from CD field outbreaks, to experimentally infect turkeys to evaluate local (skin and muscle) and systemic (spleen) pathological and immunological responses. Results showed that while all three strains produced an acute disease, Str. A1 and B1 caused significantly higher mortality when compared to Str. C1. Gross and histopathology evaluation showed that birds infected with Str. A1 and B1 had severe inflammatory, edematous, granulomatous and necrotic lesions in the skin, muscle and spleen, while these lesions produced by Str. C1 were relatively less severe and mostly confined to skin and/or muscle. Immune gene expression in these tissues showed that Str. B1-infected birds had significantly higher expression of interleukin (IL)-1ß, IL-6 and interferon (IFN)γ genes compared to uninfected control, suggesting a robust inflammatory response both locally as well as systemically. The transcription of IL-1ß and IFNγ in the muscle or spleen of Str. A1-infected birds and IL-1ß in the skin of Str. C1-infected group was also significantly higher than control. Additionally, Str. A1 or B1-infected groups also had significantly higher IL-4 transcription in these tissues, while birds infected with all three strains developed C. septicum-specific serum antibodies. Furthermore, splenic cellular immunophenotyping in the infected turkeys showed a marked reduction in CD4+ cells. Collectively, it can be inferred that host responses against C. septicum involve an acute inflammatory response along with antibody production and that the disease severity seem to depend on the strain of C. septicum involved in CD in turkeys.

Immunization of turkeys with Clostridium septicum alpha toxin-based recombinant subunit proteins can confer protection against experimental Clostridial dermatitis.

Clostridial dermatitis (CD), caused by Clostridium septicum, is an emerging disease of increasing economic importance in turkeys. Currently, there are no effective vaccines for CD control. Here, two non-toxic domains of C. septicum alpha toxin, namely ntATX-D1 and ntATX-D2, were identified, cloned, and expressed in Escherichia coli as recombinant subunit proteins to investigate their use as potential vaccine candidates. Experimental groups consisted of a Negative control (NCx) that did not receive C. septicum challenge, while the adjuvant-only Positive control (PCx), ntATX-D1 immunization (D1) and ntATX-D2 immunization (D2) groups received C. septicum challenge. Turkeys were immunized subcutaneously with 100 µg of protein at 7, 8 and 9 weeks of age along with an oil-in-water nano-emulsion adjuvant, followed by C. septicum challenge at 11 weeks of age. Results showed that while 46.2% of birds in the PCx group died post-challenge, the rate of mortality in D1- or D2-immunization groups was 13.3%. The gross and histopathological lesions in the skin, muscle and spleen showed that the disease severity was highest in PCx group, while the D2-immunized birds had significantly lower lesion scores when compared to PCx. Gene expression analysis revealed that PCx birds had significantly higher expression of pro-inflammatory cytokine genes in the skin, muscle and spleen than the NCx group, while the D2 group had significantly lower expression of these genes compared to PCx. Peripheral blood cellular analysis showed increased frequencies of activated CD4+ and/or CD8+ cells in the D1 and D2-immunized groups. Additionally, the immunized turkeys developed antigen-specific serum IgY antibodies. Collectively, these findings indicate that ntATX proteins, specifically the ntATX-D2 can be a promising vaccine candidate for protecting turkeys against CD and that the protection mechanisms may include downregulation of C. septicum-induced inflammation and increased CD4+ and CD8+ cellular activation.

Vaccination of turkeys with Clostridium septicum bacterin-toxoid: evaluation of protection against clostridial dermatitis.

Clostridial dermatitis is an acute disease causing high mortality in turkeys. Both Clostridium septicum and Clostridium pefringens have been isolated from these cases; however, reports from several diagnostic laboratories indicate an increased isolation rate of C septicum compared with C. perfringens from cases of clostridial dermatitis in recent years. Previous studies suggested C. septicum was more potent than C. perfringens in causing clostridial dermatitis in turkeys. The objective of this study was to develop and evaluate the use of a C. septicum bacterin-toxoid to control clostridial dermatitis in turkeys. A C. septicum bacterin-toxoid was prepared and was initially tested in 6-wk-old commercial turkeys under laboratory conditions for its safety and efficacy. Subsequently, the bacterin-toxoid was evaluated for use in commercial turkey farms with a consistent history of clostridial dermatitis. Birds in the field were vaccinated subcutaneously once at 6 wk of age with C. septicum bacterin-toxoid, and then mortality in both vaccinated and unvaccinated groups was recorded and compared. Blood samples from birds in both groups were examined using ELISA to detect antibody response to the C. septicum toxoid. The C. septicum bacterin-toxoid was found to be safe and to elicit antibodies against the toxoid. In vaccinated commercial turkeys, control of clostridial dermatitis was achieved via antibiotic use and clostridial dermatitis mortality was significantly reduced compared with that of birds in the unvaccinated group. The C. septicum bacterin-toxoid seems to be a valuable tool for the turkey industry to reduce losses due to clostridial dermatitis.

Immune Response Evaluation in Commercial Turkeys Affected with Clostridial Dermatitis.

Clostridial dermatitis (CD), caused by Clostridium septicum and Clostridium perfringens, is an economically important emerging disease of turkeys characterized by sudden deaths and necrotic dermatitis. Immune responses in CD-affected commercial turkeys are poorly understood. In the present study, C. septicum was isolated from CD-affected commercial turkeys during a recent outbreak, and the tissues (skin, muscle, and spleen) were collected and analyzed for immune gene expression, along with samples from clinically healthy birds. The results showed that CD-affected turkeys had significantly higher levels of IL-1ß, IL-6, IFNγ, and iNOS transcripts in the skin, muscle, and spleen tissues compared to healthy birds. Affected turkeys also had a significantly elevated transcription of toll-like receptor (TLR21) gene in the skin and spleen tissues, suggesting a role for this receptor in the immune recognition. The expression of IL-4 and IL-13 genes in the spleen and muscle was also significantly higher in the affected birds. Additional birds from the same affected and healthy farms examined for serology revealed that the CD-affected turkeys had significantly higher levels of serum IgM and IgY antibodies. Furthermore, in vitro stimulation of MQ-NCSU macrophages with C. septicum led to a significant transcriptional upregulation of IL-1ß and IFNγ genes, while the IL-10 gene expression was downregulated. The surface expression of MHC-II protein and cellular production of nitric oxide were also significantly increased in the C. septicum-stimulated macrophages, indicating cellular activation. Collectively, our findings suggest that the host responses in CD-affected turkeys involve a robust inflammatory response as well as a response mediated by IL4/IL-13 cytokines that may aid in antibody-mediated immunity.

Evaluación de la respuesta inmune en pavos comerciales afectados por dermatitis clostridial. La dermatitis clostridial (CD), causada por Clostridium septicum y Clostridium perfringens, es una enfermedad emergente económicamente importante de los pavos caracterizada por muerte súbitas y dermatitis necrótica. Se conoce poco acerca de las respuestas inmunitarias en pavos comerciales afectados por dermatitis clostridial. En el presente estudio, se aisló C. septicum de pavos comerciales afectados por dermatitis clostridial durante un brote reciente, y los tejidos (piel, músculo y bazo) se recolectaron y analizaron para determinar la expresión de genes inmunitarios junto con muestras de aves clínicamente sanas. Los resultados mostraron que los pavos afectados por dermatitis clostridial tenían niveles significativamente más altos de transcritos de IL-1ß, IL-6, IFNγ, and iNOS en los tejidos de la piel, los músculos y el bazo en comparación con las aves sanas. Los pavos afectados también tenían una transcripción significativamente elevada del gene del receptor tipo toll (TLR21) en los tejidos de la piel y el bazo, lo que sugiere un papel de este receptor en el reconocimiento inmunitario. La expresión de los genes IL-4 e IL-13 en el bazo y el músculo también fue significativamente mayor en las aves afectadas. Aves adicionales de las mismas granjas afectadas y sanas que fueron examinadas por serología revelaron que los pavos afectados por dermatitis clostridial tenían niveles significativamente más altos de anticuerpos séricos IgM e IgY. Además, la estimulación in vitro de los macrófagos MQ-NCSU con C. septicum condujo a una regulación transcripcional significativamente al alza de los genes IL-1ß and IFNγ, mientras que la expresión del gene IL-10 se reguló a la baja. La expresión superficial de la proteína MHC-II y la producción celular de óxido nítrico también aumentaron significativamente en los macrófagos estimulados por C. septicum, lo que indica activación celular. Colectivamente, estos hallazgos sugieren que las respuestas del huésped en pavos afectados por dermatitis clostridial implican una respuesta inflamatoria robusta, así como una respuesta mediada por citoquinas IL4/IL-13 que pueden ayudar en la inmunidad mediada por anticuerpos.

Effect of antimicrobial administration on fecal microbiota of critically ill dogs: dynamics of antimicrobial resistance over time.

BACKGROUND: Multidrug resistance in companion animals poses significant risks to animal and human health. Prolonged antimicrobial drug (AMD) treatment in animals is a potential source of selection pressure for antimicrobial resistance (AMR) including in the gastrointestinal microbiota. We performed a prospective study of dogs treated for septic peritonitis, pyometra, or bacterial pneumonia and collected repeated fecal samples over 60 days. Bacterial cultures and direct molecular analyses of fecal samples were performed including targeted resistance gene profiling. RESULTS: Resistant Escherichia coli increased after 1 week of treatment (D1:21.4% vs. D7:67.9% P < 0.001) and returned to baseline proportions by D60 (D7:67.9% vs D60:42.9%, P = 0.04). Dogs with septic peritonitis were hospitalized significantly longer than those with pneumonia or pyometra. Based on genetic analysis, Simpson's diversity index significantly decreased after 1 week of treatment (D1 to D7, P = 0.008), followed by a gradual increase to day 60 (D1 and D60, P = 0.4). Detection of CTX-M was associated with phenotypic resistance to third-generation cephalosporins in E. coli (OR 12.1, 3.3-68.0, P < 0.001). Lincosamide and macrolide-resistance genes were more frequently recovered on days 14 and 28 compared to day 1 (P = 0.002 and P = 0.004 respectively). CONCLUSION: AMR was associated with prescribed drugs but also developed against AMDs not administered during the study. Companion animals may be reservoirs of zoonotic multidrug resistant pathogens, suggesting that veterinary AMD stewardship and surveillance efforts should be prioritized.

Genomics accurately predicts antimicrobial resistance in Staphylococcus pseudintermedius collected as part of Vet-LIRN resistance monitoring.

Whole-genome sequencing (WGS) has changed our understanding of bacterial pathogens, aiding outbreak investigations and advancing our knowledge of their genetic features. However, there has been limited use of genomics to understand antimicrobial resistance of veterinary pathogens, which would help identify emerging resistance mechanisms and track their spread. The objectives of this study were to evaluate the correlation between resistance genotypes and phenotypes for Staphylococcus pseudintermedius, a major pathogen of companion animals, by comparing broth microdilution antimicrobial susceptibility testing and WGS. From 2017-2019, we conducted antimicrobial susceptibility testing and WGS on S. pseudintermedius isolates collected from dogs in the United States as a part of the Veterinary Laboratory Investigation and Response Network (Vet-LIRN) antimicrobial resistance monitoring program. Across thirteen antimicrobials in nine classes, resistance genotypes correlated with clinical resistance phenotypes 98.4 % of the time among a collection of 592 isolates. Our findings represent isolates from diverse lineages based on phylogenetic analyses, and these strong correlations are comparable to those from studies of several human pathogens such as Staphylococcus aureus and Salmonella enterica. We uncovered some important findings, including that 32.3 % of isolates had the mecA gene, which correlated with oxacillin resistance 97.0 % of the time. We also identified a novel rpoB mutation likely encoding rifampin resistance. These results show the value in using WGS to assess antimicrobial resistance in veterinary pathogens and to reveal putative new mechanisms of resistance.

Role of Clostridium perfringens and Clostridium septicum in causing turkey cellulitis.

The role of Clostridium perfringens and Clostridium septicum in the development of cellulitis and mortality in turkey poults was examined. Studies were done in turkeys of two age groups: 3-wk-old and 7-wk-old turkey poults. The effect of varying doses of C. perfringens and C. septicum in reproducing cellulitis lesions and mortality in turkeys was investigated. Both in vitro and in vivo assays were conducted to study their toxic and biologic activities. Clostridium septicum spore culture was found to be more potent than that of C. perfringens in both in vitro assays, such as the hemolysis test, and in vivo assays in mice and turkeys. Both C. perfringens and C. septicum spore cultures were found to be capable of inducing cellulitis lesions and mortality in turkey poults when inoculated by subcutaneous route. Histopathology examination of affected tissues revealed a "moth-eaten appearance, with abundant growth of C. perfringens and C. septicum in the sarcomeres of muscle tissues and in the subcutaneous tissues. However, C. septicum was found to be more potent than C. perfringens in causing cellulitis lesions and mortality in turkeys. Three-week-old poults were found to be less susceptible than 7-wk-old poults in the development of cellulitis lesions and mortality after inoculation with either spore cultures of C. perfringens or C. septicum. The results of the current study suggest that although C. septicum is more potent in causing cellulitis lesions and mortality, infection with either C. septicum or C. perfringens can cause cellulitis lesions and mortality in turkeys.

Characterization of a novel Mycoplasma cynos real-time PCR assay.

Mycoplasma cynos is recognized as an emerging causative pathogen of canine infectious respiratory disease (CIRD) worldwide. We developed a new open-source real-time PCR (rtPCR) assay for M. cynos that performs well under standard rtPCR conditions. Primers and probes were designed to target the M. cynos tuf gene. Reaction efficiencies for the M. cynos tuf gene assay on 2 platforms were based on amplification of standard curves spanning 8 orders of magnitude: ABI 7500 platform, 94.3-97.9% (r2 ≥ 0.9935); QuantStudio OpenArray platform, 119.1-122.5% (r2 = 0.9784). The assay performed very well over a range of template input, from 109 copies to the lower limit of quantification at 4 copies of the M. cynos genome on the ABI 7500 platform. Diagnostic performance was estimated by comparison with an in-house legacy assay on clinical specimens as well as testing isolates that were characterized previously by intergenic spacer region (ISR) sequencing. Exclusivity was established by testing 12 other Mycoplasma species. To substantiate the high specificity of the M. cynos tuf gene assay, sequence confirmation was performed on ISR PCR amplicons obtained from clinical specimens. One ISR amplicon sequence revealed M. mucosicanis rather than M. cynos. The complete protocol of the newly developed M. cynos tuf assay is provided to facilitate assay harmonization.

Geography Shapes the Population Genomics of Salmonella enterica Dublin.

Salmonella enterica serotype Dublin (S. Dublin) is a bovine-adapted serotype that can cause serious systemic infections in humans. Despite the increasing prevalence of human infections and the negative impact on agricultural processes, little is known about the population structure of the serotype. To this end, we compiled a manually curated data set comprising of 880 S. Dublin genomes. Core genome phylogeny and ancestral state reconstruction revealed that region-specific clades dominate the global population structure of S. Dublin. Strains of S. Dublin in the UK are genomically distinct from US, Brazilian, and African strains. The geographical partitioning impacts the composition of the core genome as well as the ancillary genome. Antibiotic resistance genes are almost exclusively found in US genomes and are mediated by an IncA/C2 plasmid. Phage content and the S. Dublin virulence plasmid were strongly conserved in the serotype. Comparison of S. Dublin to a closely related serotype, S. enterica serotype Enteritidis, revealed that S. Dublin contains 82 serotype specific genes that are not found in S. Enteritidis. Said genes encode metabolic functions involved in the uptake and catabolism of carbohydrates and virulence genes associated with type VI secretion systems and fimbria assembly respectively.

LEPTOSPIROSIS IN URBAN AND SUBURBAN AMERICAN BLACK BEARS ( URSUS AMERICANUS) IN WESTERN NORTH CAROLINA, USA.

American black bear ( Ursus americanus) populations in North Carolina, US have recovered significantly in recent decades and now occupy much of western North Carolina, including urban-suburban areas. We used the black bear as a potential sentinel for leptospirosis, a bacterial zoonotic disease caused by Leptospira spp., which is maintained by domestic and wild mammals. We determined whether Leptospira spp. were present across a gradient of housing densities in the urban and suburban black bear population in and around Asheville, North Carolina using serologic and molecular surveys. We collected blood from captured black bears ( n=94) and kidneys and bladders from carcasses ( n=19). We tested a total of 96 (47 females, 47 males, and 2 unknown) serum samples by microscopic agglutination test (MAT) and had positive results (titer >1:100) for L. kirschneri serovar Grippotyphosa (L. Grippotyphosa) in 4 females (8%) and 5 males (10%). No other serovars showed elevated titers in MAT. We tested a total of 125 samples using PCR ( n=96 serum, n=20 kidney, and n=9 bladders) and obtained positive results from one serum (1%), one kidney (5%), and one bladder (11%). The presence of Leptospira spp. in black bears occupying an urban and suburban landscape may indicate a more extensive occurrence of the bacteria among animals in the study region because black bears are the top carnivore in that ecosystem. Potential threats of widespread contamination during natural events such as flood or drought must be considered.

Frequent human-poultry interactions and low prevalence of Salmonella in backyard chicken flocks in Massachusetts.

The backyard chicken (BYC) movement in the USA has increased human contact with poultry and subsequently, human contact with the pathogen Salmonella. However, to date, there have been few studies assessing prevalence of Salmonella in backyard flocks, despite the known public health risk this zoonotic bacterium poses. The objective of this study was to characterize human-BYC interactions and assess the prevalence of Salmonella among BYC flocks. We interviewed 50 BYC owners using a structured questionnaire to determine flock and household characteristics that facilitate contact with BYC and that may be associated with Salmonella in the BYC environment. Composite faecal material, cloacal swabs and dust samples from 53 flocks housed on 50 residential properties in the Greater Boston, Massachusetts area were tested for Salmonella using standard culture techniques and confirmed using Matrix-Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometer. Microbroth dilution and whole genome sequencing were used to determine phenotypic and genotypic resistance profiles, respectively, and sequence results were used to determine multilocus sequencing type. No owners self-reported a diagnosis of salmonellosis in the household. Over 75% of a subset of owners reported that they and their children consider BYC pets. This perception is evident in how owners reported interacting with their birds. Salmonella enterica subspecies enterica serotype Kentucky ST152 (serogroup C)-a strain not commonly associated with human infection-was confirmed in one flock, or 2% of tested flocks, and demonstrated resistance to tetracycline and streptomycin. We detected Salmonella at low prevalence in BYC. Further study of the health effects of exposure to zoonotic gastrointestinal pathogens such as Salmonella among families with BYC is warranted.

Atypical Dermatophytosis in 12 North American Porcupines (Erethizon dorsatum) from the Northeastern United States 2010-2017.

Twelve wild North American porcupines (Erethizon dorsatum) out of a total of 44 of this species examined in an 8-year period were diagnosed with dermatopathies while being cared for at two wildlife rehabilitation clinics. Biopsy and necropsy were performed on seven and five animals, respectively. Atypical dermatophytosis was diagnosed in all cases. Lesions consisted of diffuse severe epidermal hyperkeratosis and mild hyperplasia with mild lymphoplasmacytic dermatitis and no folliculitis. Dermatophytes were noted histologically as hyphae and spores in hair shafts, and follicular and epidermal keratin. Trichophyton sp. was grown in 5/6 animals where culture was performed, with a molecular diagnosis of Arthroderma benhamiae/Trichophyton mentagrophytes in these five cases. Metagenomic analysis of formalin-fixed paraffin-embedded tissue samples from three cases identified fungi from 17 orders in phyla Basidiomycota and Ascomycota. Alteration of therapy from ketaconazole, which was unsuccessful in four out of five early cases, to terbinafine or nitraconazole led to the resolution of disease and recovery to release in four subsequent animals. In all, six animals were euthanized or died due to dermatopathy, no cases resolved spontaneously, and six cases were resolved with therapy. The work we present demonstrates an atypical lesion and anatomical distribution due to dermatophytosis in a series of free-ranging wild porcupines and the successful development of novel techniques for extracting and sequencing nucleic acids from fungus in archival formalin-fixed paraffin-embedded animal tissue.

Comparative pathogenicity of early and recent isolates of avian metapneumovirus subtype C in turkeys.

The objective of the present study was to compare the pathogenicity of early and recent isolates of avian metapneumovirus subtype-C (aMPV-C) in turkeys. Two-week-old turkeys were inoculated with early and recent isolates of aMPV-C. Clinical signs were monitored. Tissues were examined for viral ribonucleic acid (RNA), lesions, and viral antigen by reverse transcription-polymerase chain reaction (RT-PCR), histopathology and immunohistochemistry, respectively. Birds infected with the recent isolate had higher clinical sign scores than those infected with the early isolate. Only the recent isolate produced a multifocal loss of cilia in the nasal turbinate of infected birds. Immunohistochemistry revealed intense staining of aMPV antigen in turbinate and trachea of birds infected with the recent isolate. The findings indicate that the recent isolate produced more severe clinical signs and lesions in turkeys compared to the early isolate. The recent isolate could be ideal for the development of a challenge model for aMPV infection in turkeys.

Application of polymerase chain reaction fingerprinting to differentiate Ornithobacterium rhinotracheale isolates.

Ornithobacterium rhinotracheale (ORT) is an infectious respiratory pathogen of chickens, turkeys, and wild birds. There are 18 serotypes of ORT reported worldwide. In this study, enterobacterial repetitive intergenic consensus (ERIC) polymerase chain reaction and random amplified polymorphic DNA assay with Universal M13 primer-based fingerprinting techniques were investigated for their ability to differentiate ORT isolates. The authors examined 50 field isolates and 8 reference strains of ORT for their genetic differences. The fingerprint patterns were compared with serotyping results of ORT by the agar gel precipitation test. M13 fingerprinting revealed different patterns for 6 reference serotypes of ORT that were tested, namely, C, D, E, I, J, and K. Ornithobacterium rhinotracheale reference serotypes A and F yielded indistinguishable fingerprints with M13 fingerprinting. The ERIC 1R technique discerned only 5 of the 8 reference serotypes of ORT. Distinct fingerprints were also found within the ORT serotypes with both techniques. From 58 isolates of ORT that were fingerprinted belonging to 8 ORT serotypes, 10 different fingerprints were obtained with M13 fingerprinting and 6 different fingerprints were obtained with ERIC 1R fingerprinting. M13 fingerprinting technique was found to be more discriminative in differentiating ORT isolates than the ERIC 1R fingerprinting technique. These results suggest that fingerprinting techniques may be a more discerning tool for characterizing ORT isolates than the serological test using the agar gel precipitation test. This fingerprinting technique could potentially be a valuable tool in identifying an isolate from a clinical outbreak of ORT infection for development of an autogenous vaccine.

Discrimination of Streptococcus equi subsp. equi and Streptococcus equi subsp. zooepidemicus using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Accurate and timely identification of infectious etiologies is of great significance in veterinary microbiology, especially for critical diseases such as strangles, a highly contagious disease of horses caused by Streptococcus equi subsp. equi. We evaluated a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) platform for use in species- and subspecies-level identification of S. equi isolates from horses and compared it with an automated biochemical system. We used 25 clinical isolates each of S. equi subsp. equi and S. equi subsp. zooepidemicus. Using the MALDI-TOF MS platform, it was possible to correctly identify all 50 isolates to the species level. Unique mass peaks were identified in the bacterial peptide mass spectra generated by MALDI-TOF MS, which can be used for accurate subspecies-level identification of S. equi. Mass peaks (mass/charge, m/ z) 6,751.9 ± 1.4 (mean ± standard deviation) and 5,958.1 ± 1.3 were found to be unique to S. equi subsp. equi and S. equi subsp. zooepidemicus, respectively. The automated biochemical system correctly identified 47 of 50 of the isolates to the species level as S. equi, whereas at the subspecies level, 24 of 25 S. equi subsp. equi isolates and 22 of 25 S. equi subsp. zooepidemicus isolates were correctly identified. Our results indicate that MALDI-TOF MS can be used for accurate species- and subspecies-level identification of S. equi.