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Detection of Leptospira interrogans is difficult as a result of intermittent leptospiruria and brief leptospiremia. Hence, diagnosis relies heavily on serologic testing, the reference method of which is the microscopic agglutination test (MAT). In horses, clinical leptospirosis has been associated with abortion, recurrent uveitis, and sporadic cases of hepatic and renal disease. Little information exists on the seroprevalence of antibodies to L. interrogans in equids in the United States; past nationwide studies suggest that the seroprevalence in some areas is as high as 77% (reciprocal titer ≥ 100). We tested sera from 124 apparently healthy horses previously submitted for equine infectious anemia (EIA) serology using MAT for 6 serovars—Bratislava, Canicola, Grippotyphosa, Hardjo, Icterohaemorrhagiae, and Pomona. When using a reciprocal MAT titer cutoff of ≥ 100, 102 of 124 (82%) of the samples were positive for at least one serovar. Seropositivity was significantly associated with increasing age. Query of specimens from clinical cases submitted to the Colorado State University Veterinary Diagnostic Laboratory for MAT since 2010 indicated significantly greater seroprevalence ( p = 0.015) of pathogenic serovar Pomona in clinical cases compared to sera submitted from healthy equids for routine EIA testing. Information from our diagnostic laboratory submission forms also suggests a correlation between uveitis or other ophthalmic problems and serovar Pomona.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in 2019 and has become a major global pathogen in an astonishingly short period of time. The emergence of SARS-CoV-2 has been notable due to its impacts on residents in long-term care facilities (LTCFs). LTCF residents tend to possess several risk factors for severe outcomes of SARS-CoV-2 infection, including advanced age and the presence of comorbidities. Indeed, residents of LTCFs represent approximately 40% of SARS-CoV-2 deaths in the United States. Few studies have focused on the prevalence and transmission dynamics of SARS-CoV-2 among LTCF staff during the early months of the pandemic, prior to mandated surveillance testing. To assess the prevalence and incidence of SARS-CoV-2 among LTCF staff, characterize the extent of asymptomatic infections, and investigate the genomic epidemiology of the virus within these settings, we sampled staff for 8 to 11 weeks at six LTCFs with nasopharyngeal swabs from March through June of 2020. We determined the presence and levels of viral RNA and infectious virus and sequenced 54 nearly complete genomes. Our data revealed that over 50% of infections were asymptomatic/mildly symptomatic and that there was a strongly significant relationship between viral RNA (vRNA) and infectious virus, prolonged infections, and persistent vRNA (4+ weeks) in a subset of individuals, and declining incidence over time. Our data suggest that asymptomatic SARS-CoV-2-infected LTCF staff contributed to virus persistence and transmission within the workplace during the early pandemic period. Genetic epidemiology data generated from samples collected during this period support that SARS-CoV-2 was commonly spread between staff within an LTCF and that multiple-introduction events were less common. IMPORTANCE Our work comprises unique data on the characteristics of SARS-CoV-2 dynamics among staff working at LTCFs in the early months of the SARS-CoV-2 pandemic prior to mandated staff surveillance testing. During this time period, LTCF residents were largely sheltering-in-place. Given that staff were able to leave and return daily and could therefore be a continued source of imported or exported infection, we performed weekly SARS-CoV-2 PCR on nasal swab samples collected from this population. There are limited data from the early months of the pandemic comprising longitudinal surveillance of staff at LTCFs. Our data reveal the surprisingly high level of asymptomatic/presymptomatic infections within this cohort during the early months of the pandemic and show genetic epidemiological analyses that add novel insights into both the origin and transmission of SARS-CoV-2 within LTCFs.

Steller sea lions (SSLs) Eumetopias jubatus experienced a population decline in the 1960s, leading to the listing of the western stock as endangered and the eastern stock as threatened under the US Endangered Species Act. A decrease of births in the western stock beginning in the late 1960s indicates that reproductive failure may have contributed to the decline. We evaluated the role pathogens play in spontaneous abortions, premature births and neonatal deaths in SSLs. Archived tissues from carcasses (n = 19) collected in Alaska from 2002 to 2015 were tested by PCR for Coxiella burnetii, Brucella spp., Chlamydia and morbilliviruses. Animals examined included 47% premature pups, 32% aborted fetuses, 11% neonates and 11% intrauterine fetuses. Gross necropsy and histology findings were summarized in the context of the PCR findings. Tissues were negative for Chlamydia and C. burnetii. Brucella spp. were detected in the lung tissues of 3 animals, including 1 positive for the ST27 strain, the first detection of Brucella spp. DNA in SSLs. Phocine distemper virus was detected in 3 animals in 2 skin lesions and 1 placenta by hemi-nested diagnostic qRT-PCR. Both skin and the placental lesions had vesiculoulcerative changes, and 1 skin lesion contained inclusion bodies in syncytia and upon histologic examination, suggesting that the lesions may be associated with an infection reminiscent of phocine distemper virus, the first in SSLs. We highlight the continuing need for disease surveillance programs to improve our understanding of the prevalence and potential population impacts of these infectious disease agents for pinnipeds in Alaskan waters.

In the United States, ~1.4 million sporadic human Salmonella enterica infections occur annually, with an estimated 6% attributable to reptile exposure. Detection of Salmonella in reptiles can be challenging given the limitations among detection methods. We evaluated sampling and detection methods for S. enterica in a cross-sectional study of reptilian patients ( n = 45) over the course of 13 mo. Two sampling methods (cloacal swabs, electrostatic cloth body-feet samples) and 3 detection methods (enriched culture, lateral flow immunoassay [LFI], real-time PCR) were compared using McNemar and Fisher exact tests. Results varied by species, sample type, and detection method. In total, 14 of 45 (33%) patients were positive by culture, 10 of 45 (22%), and/or 13 of 45 (29%) by rtPCR. Among rtPCR-positive results, cloacal swabs (12 of 45 [27%]) resulted in a higher detection than body-feet wipes (4 of 45 [9%]; p = 0.01). Among culture-positive results, shedding was most commonly detected after additional incubation at room temperature when testing cloacal swabs (9 of 45 [20%]). However, there was significant disagreement between sampling methods (cloacal vs. body-feet; p = 0.03). No samples were positive by LFI. In general, cloacal swabs yielded the highest test-positive rates, irrespective of testing method. Our study highlights the importance of using detection methods optimized for the sample being tested.

A comprehensive understanding of common diseases of backyard poultry flocks is important to providing poultry health information to flock owners, veterinarians, and animal health officials. We collected autopsy reports over a 3-y period (2015–2017) from diagnostic laboratories in 8 states in the United States; 2,509 reports were collected, involving autopsies of 2,687 birds. The primary cause of mortality was categorized as infectious, noninfectious, neoplasia or lymphoproliferative disease, or undetermined. Neoplasia or lymphoproliferative disease was the most common primary diagnosis and involved 42% of the total birds autopsied; 63% of these cases were diagnosed as Marek’s disease or leukosis/sarcoma. Bacterial, parasitic, and viral organisms were commonly detected, involving 42%, 28%, and 7% of the birds autopsied, respectively, with 2 or more organisms detected in 69% of birds. Our findings demonstrate the importance of educating flock owners about disease prevention and biosecurity practices. The detection of zoonotic bacteria including paratyphoid salmonellae, Campylobacter spp., Listeria monocytogenes, and Mycobacterium avium, and the detection of lead and other heavy metals, indicate public health risks to flock owners and consumers of backyard flock egg and meat products.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in 2019 and has become a major global pathogen in an astonishingly short period of time. The emergence of SARS-CoV-2 has been notable due to its impacts on residents in long-term care facilities (LTCFs). LTCF residents tend to possess several risk factors for severe outcomes of SARS-CoV-2 infection, including advanced age and the presence of comorbidities. Indeed, residents of LTCFs represent approximately 40% of SARS-CoV-2 deaths in the United States. Few studies have focused on the prevalence and transmission dynamics of SARS-CoV-2 among LTCF staff during the early months of the pandemic, prior to mandated surveillance testing. To assess the prevalence and incidence of SARS-CoV-2 among LTCF staff, characterize the extent of asymptomatic infections, and investigate the genomic epidemiology of the virus within these settings, we sampled staff for 8 to 11 weeks at six LTCFs with nasopharyngeal swabs from March through June of 2020. We determined the presence and levels of viral RNA and infectious virus and sequenced 54 nearly complete genomes. Our data revealed that over 50% of infections were asymptomatic/mildly symptomatic and that there was a strongly significant relationship between viral RNA (vRNA) and infectious virus, prolonged infections, and persistent vRNA (4+ weeks) in a subset of individuals, and declining incidence over time. Our data suggest that asymptomatic SARS-CoV-2-infected LTCF staff contributed to virus persistence and transmission within the workplace during the early pandemic period. Genetic epidemiology data generated from samples collected during this period support that SARS-CoV-2 was commonly spread between staff within an LTCF and that multiple-introduction events were less common. IMPORTANCE Our work comprises unique data on the characteristics of SARS-CoV-2 dynamics among staff working at LTCFs in the early months of the SARS-CoV-2 pandemic prior to mandated staff surveillance testing. During this time period, LTCF residents were largely sheltering-in-place. Given that staff were able to leave and return daily and could therefore be a continued source of imported or exported infection, we performed weekly SARS-CoV-2 PCR on nasal swab samples collected from this population. There are limited data from the early months of the pandemic comprising longitudinal surveillance of staff at LTCFs. Our data reveal the surprisingly high level of asymptomatic/presymptomatic infections within this cohort during the early months of the pandemic and show genetic epidemiological analyses that add novel insights into both the origin and transmission of SARS-CoV-2 within LTCFs.

Isolation and cultivation of wild-type viruses in model organism cells or tissues is standard practice in virology. Oftentimes, the virus host species is distantly related to the species from which the culture system was developed. Thus, virus culture in these tissues and cells basically constitutes a host jump, which can lead to genomic changes through genetic drift and/or adaptation to the culture system. We directly sequenced 70 avian influenza virus (Orthomyxoviridae) genomes from oropharyngeal/cloacal swabs collected from wild bird species and paired virus isolates propagated from the same samples following isolation in specific-pathogen-free embryonated chicken eggs. The data were analyzed using population genetic approaches including evaluation of single nucleotide polymorphism (SNP) frequencies and divergence with pooled-sequencing analyses, consensus sequence placement in neighbor-joining trees, and haplotype reconstruction and networks. We found that propagation of virus in eggs leads to skewed SNP mutation spectra with some SNPs going to fixation. Both synonymous and nonsynonmous SNP frequencies shifted. We found multiple consensus sequences that differed between the swabs and the isolates, with some sequences from the same sample falling into divergent genetic clusters. Twenty of 23 coinfections detected had different dominant subtypes following virus isolation, thus sequences from both the swab and isolate were needed to obtain full subtype data. Haplotype networks revealed haplotype frequency shifts and the appearance or loss of low-frequency haplotypes following isolation. The results from this study revealed that isolation of wild bird avian influenza viruses in chicken eggs leads to skewed populations that are different than the input populations. Consensus sequence changes from virus isolation can lead to flawed phylogenetic inferences, and subtype detection is biased. These results suggest that for genomic studies of wild bird influenza viruses the biological field should move away from chicken egg isolation towards directly sequencing the virus from host samples.

Background Plague caused by Yersinia pestis is a highly infectious and potentially fatal zoonotic disease that can be spread by wild and domestic animals. In endemic areas of the northern hemisphere plague typically cycles from March to October, when flea vectors are active. Clinical forms of disease include bubonic, septicemic, and pneumonic plague. All clinical forms are uncommon in dogs and the pneumonic form is exceedingly rare. Case presentation Two mixed breed young-adult male domestic dogs presented to Colorado veterinarians with fever and vague signs that progressed to hemoptysis within 24 h. Case 1 presented in June 2014, while Case 2 occurred in December 2017. Thoracic radiography of Case 1 and 2 revealed right dorsal and right accessory lobe consolidation, respectively. In Case 1 initial differential diagnoses included pulmonary contusion due to trauma or diphacinone toxicosis. Case 1 was euthanized ~ 24 h post presentation due to progressive dyspnea and hemoptysis. Plague was confirmed 9 days later, after the dog’s owner was hospitalized with pneumonia. Case 2 was treated as foreign body/aspiration pneumonia and underwent lung lobectomy at a veterinary teaching hospital. Case 2 was euthanized after 5 days of hospitalization when bacterial culture of the excised lobe yielded Yersinia pestis. Both dogs had severe diffuse necrohemorrhagic and suppurative pneumonia at post mortem examination. Conclusions Both dogs were misdiagnosed due to the atypical lobar presentation of an extremely rare form of plague in a species that infrequently succumbs to clinical disease. Presentation outside of the typical transmission period of plague was also a factor leading to delayed diagnosis in Case 2. Erroneous identification by automated bacterial identification systems was problematic in both cases. In endemic areas, plague should be ruled out early in febrile dogs with acute respiratory signs, hemoptysis, lobar or diffuse pathology, and potential for exposure, regardless of season. Seasonal and geographic distributions of plague may shift with climate change, so vigilance by primary care veterinarians is warranted. Timely submission of samples to a veterinary diagnostic laboratory could expedite accurate diagnosis and reduce potential for human and domestic animal exposure.

Introduction Multidrug resistance (MDR) is a serious issue prevalent in various agriculture-related foodborne pathogens including Salmonella enterica (S. enterica) Typhimurium. Class I integrons have been detected in Salmonella spp. strains isolated from food producing animals and humans and likely play a critical role in transmitting antimicrobial resistance within and between livestock and human populations. Objective The main objective of our study was to characterize class I integron presence to identify possible integron diversity among and between antimicrobial resistant Salmonella Typhimurium isolates from various host species, including humans, cattle, swine, and poultry. Methods An association between integron presence with multidrug resistance was evaluated. One hundred and eighty-three S. Typhimurium isolates were tested for antimicrobial resistance (AMR). Class I integrons were detected and sequenced. Similarity of AMR patterns between host species was also studied within each integron type. Results One hundred seventy-four (95.1%) of 183 S.Typhimurium isolates were resistant to at least one antimicrobial and 82 (44.8%) were resistant to 5 or more antimicrobials. The majority of isolates resistant to at least one antimicrobial was from humans (45.9%), followed by swine (19.1%) and then bovine (16.9%) isolates; poultry showed the lowest number (13.1%) of resistant isolates. Our study has demonstrated high occurrence of class I integrons in S. Typhimurium across different host species. Only one integron size was detected in poultry isolates. There was a significant association between integron presence of any size and specific multidrug resistance pattern among the isolates from human, bovine and swine. Conclusions Our study has demonstrated a high occurrence of class I integrons of different sizes in Salmonella Typhimurium across various host species and their association with multidrug resistance. This demonstration indicates that multidrug resistant Salmonella Typhimurium is of significant public health occurrence and reflects on the importance of judicious use of antimicrobials among livestock and poultry.

Background SARS-CoV-2 emerged in 2019 and has become a major global pathogen. Its emergence is notable due to its impacts on individuals residing within long term care facilities (LTCFs) such as rehabilitation centers and nursing homes. LTCF residents tend to possess several risk factors for more severe SARS-CoV-2 outcomes, including advanced age and multiple comorbidities. Indeed, residents of LTCFs represent approximately 40% of SARS-CoV- 2 deaths in the United States. Methods To assess the prevalence and incidence of SARS-CoV-2 among LTCF workers, determine the extent of asymptomatic SARS-CoV-2 infection, and provide information on the genomic epidemiology of the virus within these unique care settings, we collected nasopharyngeal swabs from workers for 8-11 weeks at six Colorado LTCFs, determined the presence and level of viral RNA and infectious virus within these samples, and sequenced 54 nearly complete genomes. Findings Our data reveal a strikingly high degree of asymptomatic/mildly symptomatic infection, a strong correlation between viral RNA and infectious virus, prolonged infections and persistent RNA in a subset of individuals, and declining incidence over time. Interpretation Our data suggest that asymptomatic SARS-CoV-2 infected individuals contribute to virus persistence and transmission within the workplace, due to high levels of virus. Genetic epidemiology revealed that SARS-CoV-2 likely spreads between staff within an LTCF. Funding Colorado State University Colleges of Health and Human Sciences, Veterinary Medicine and Biomedical Sciences, Natural Sciences, and Walter Scott, Jr. College of Engineering, the Columbine Health Systems Center for Healthy Aging, and the National Institute of Allergy and Infectious Diseases. Research in Context Evidence before this study We searched PubMed and Google Scholar on April 15, 2020 for manuscripts published in 2020 with the key words “SARS-CoV-2 OR COVID-19 AND Long- Term Care Facility AND Surveillance OR Screening. We did not restrict our search to the English language. Our search retrieved two reports of original research. The relevant publications described transmission and course of infection among residents in LTCFs. Of particular relevance was that large quantities of SARS-CoV-2 viral RNA could be detected in asymptomatic, presymptomatic and symptomatic residents, providing early evidence of the heterogeneity of infection characteristics among residents at LTCFs. A significant number of LTCF residents were presymptomatic with symptoms emerging 7 days after initial detection of viral RNA, indicating a longer than expected latency period. Therefore, symptomatic screening for early detection and resultant mitigation response was likely to be ineffective in preventing transmission among residents of LTCFs. There were no reports involving longitudinal surveillance testing of LTCF staff. Added value of this study While prior studies reported results of facility-wide (residents and staff) testing for SARS-CoV-2 and describe transmission dynamics among residents of LTCFs, no prior data was available describing the longitudinal characteristics of SARS-CoV-2 dynamics among staff working at LTCFs during a time period where “shelter-in-place” public guidance was in effect. During this time period, LTCF residents were largely isolated, however staff (those with both direct care and those without direct contact) were permitted to leave and return to work daily. We were therefore interested in this broad staff cohort specifically because they represent a significant and ongoing potential source of transmission within LTCFs. RT-qPCR testing for SARS-CoV-2 was performed weekly on 544 staff in six LTCFs over an 8-11-week period. Symptom data were collected and site-specific prevalence at study onset and incidence rate over time were calculated to explore the influence of identifying and removing asymptomatic SARS-CoV-2-infected individuals from the workplace. Implications of all the available evidence Our results document a surprising degree of asymptomatic/mildly symptomatic infection among apparently healthy staff, and extreme variation in SARS-CoV-2 prevalence and incidence among staff between different facilities. Plaque assay revealed a strong relationship between vRNA and infectious virus in nasopharyngeal swab material, indicating the asymptomatic or mildly symptomatic individuals are infectious. Moreover, phylogenetic analysis of SARS-CoV-2 sequences collected from LTCF staff suggest that the predominant transmission pattern is between staff members within facilities, and that individual unrelated community import events are less common. Finally, decreasing prevalence over time within facilities where longitudinal surveillance testing was performed suggests that identifying and isolating positive staff may serve as part of an effective mitigation program to prevent or curtail transmission among staff within LTCFs.

Long-term viral archives are valuable sources of research data. Each archive can store hundreds of thousands of diverse sample types. In the current era of whole genome sequencing, archived samples become a rich source of evolutionary and epidemiological data that can span years, and even decades. However, the ability to obtain high quality viral whole genome sequences from samples of various types, age, and quality is inconsistent. A minimum quality threshold that helps predict the best success of obtaining high quality genomic sequences for both recent and archived samples is highly valuable. Real-time reverse transcription PCR (rrt-PCR) and droplet digital PCR (ddPCR) are useful tools to evaluate nucleic acid integrity. We hypothesized that diagnostic rrt-PCR and ddPCR data for avian influenza virus (AIV) can predict viral whole genome sequencing success. To test this hypothesis we used RNA extracted from cloacal and oropharyngeal swabs stored in the USDA-APHIS National Wildlife Disease Program Wildlife Tissue Archive. We determined that a specific rrt-PCR Cq value or ddPCR copies/μL resulted in recovery of complete sequences of all eight AIV gene segments. We used logistic regression to estimate probabilities of whole genome recovery at 0.95 (Cq = 15, copies/μL = 49,350), 0.75 (Cq = 24, copies/μL = 16,800), 0.50 (Cq = 29, copies/μL =

This cross-sectional study evaluates self-reported behaviors and cases of SARS-CoV-2 infection among essential in-person employees during the first 6 months of the COVID-19 pandemic at a US institution of higher education., Key Points Question What was the prevalence of SARS-CoV-2 infection and associated protective behaviors among essential employees at an institution of higher education during the first 6 months of the COVID-19 pandemic in the US? Findings In this cross-sectional study of 508 essential employees, no cases of SARS-CoV-2 infection were verified by quantitative reverse transcriptase–polymerase chain reaction, and only 2 participants had measurable seroreactive IgG antibodies. High levels of handwashing and mask wearing were reported at work and outside work, and social distancing was reported significantly less often at work than outside work. Meaning The findings suggest that compliance with protective behaviors both at work and outside work may be commensurate with the safe operation of complex work environments during a pandemic., Importance Detailed analysis of infection rates paired with behavioral and employee-reported risk factors is vital to understanding how transmission of SARS-CoV-2 infection may be exacerbated or mitigated in the workplace. Institutions of higher education are heterogeneous work units that supported continued in-person employment during the COVID-19 pandemic, providing a test site for occupational health evaluation. Objective To evaluate the association between self-reported protective behaviors and prevalence of SARS-CoV-2 infection among essential in-person employees during the first 6 months of the COVID-19 pandemic in the US. Design, Setting, and Participants This cross-sectional study was conducted from July 13 to September 2, 2020, at an institution of higher education in Fort Collins, Colorado. Employees 18 years or older without symptoms of COVID-19 who identified as essential in-person workers during the first 6 months of the pandemic were included. Participants completed a survey, and blood and nasal swab samples were collected to assess active SARS-CoV-2 infection via quantitative reverse transcriptase–polymerase chain reaction (qRT-PCR) and past infection by serologic testing. Exposure Self-reported practice of protective behaviors against COVID-19 according to public health guidelines provided to employees. Main Outcomes and Measures Prevalence of current SARS-CoV-2 infection detected by qRT-PCR or previous SARS-CoV-2 infection detected by an IgG SARS-CoV-2 testing platform. The frequency of protective behavior practices and essential workers’ concerns regarding contracting COVID-19 and exposing others were measured based on survey responses. Results Among 508 participants (305 [60.0%] women, 451 [88.8%] non-Hispanic White individuals; mean [SD] age, 41.1 [12.5] years), there were no qRT-PCR positive test results, and only 2 participants (0.4%) had seroreactive IgG antibodies. Handwashing and mask wearing were reported frequently both at work (480 [94.7%] and 496 [97.8%] participants, respectively) and outside work (465 [91.5%] and 481 [94.7%] participants, respectively). Social distancing was reported less frequently at work (403 [79.5%]) than outside work (465 [91.5%]) (P

Bovine tuberculosis is a chronic disease of cattle (Bos taurus) caused by the bacterium Mycobacterium bovis. Efforts have been made in the US to eradicate the disease in cattle, but spillover into wildlife and subsequent spillback have impeded progress in some states. In particular, infection in white-tailed deer (Odocoileus virginianus) has been followed by infection in cattle in some Midwestern states. Infection has also been documented in feral swine (Sus scrofa) on the Hawaiian island of Molokai and in various European countries, but no large-scale survey of antibody exposure to the bacteria has been conducted in feral swine in the US. We tested 488 sera from feral swine collected near previously documented outbreaks of bovine tuberculosis in cattle and captive cervids, in addition to 2,237 feral swine sera collected across the US from 1 October 2013 to 30 September 2014. While all but one of the samples were antibody negative, the results are important for establishing baseline negative data since feral swine are capable reservoirs and could be implicated in future outbreaks of the disease.

In late summer 2017, we observed acute, fatal cases of bovine viral diarrhea in captive Rocky Mountain bighorn sheep ( Ovis canadensis canadensis) in Colorado following use of a contaminated modified-live bluetongue virus vaccine. Following vaccination, at least 14 of 28 (50%) vaccinated bighorn sheep developed hemorrhagic diarrhea, and 6 of 28 (21%) vaccinated bighorn sheep died. Autopsy findings were predominantly necroulcerative-to-necrohemorrhagic gastrointestinal lesions. Less frequent lesions included suffusive hemorrhages of serosal surfaces of abdominal viscera, and lymphoid necrosis in gut-associated lymphoid tissues. All of the 6 bighorn sheep that died were positive on real-time PCR (rtPCR) for bovine viral diarrhea virus (BVDV) in multiple tissues. Seroconversion to BVDV-1 and immunohistochemistry for BVDV in affected tissues confirmed rtPCR results. Next-generation sequencing confirmed a match between the infecting strain of BVDV-1b and the contaminated vaccine.

Coxiella burnetii is a globally distributed zoonotic bacterial pathogen that causes abortions in ruminant livestock. In humans, an influenza-like illness results with the potential for hospitalization, chronic infection, abortion, and fatal endocarditis. Ruminant livestock, particularly small ruminants, are hypothesized to be the primary transmission source to humans. A recent Netherlands outbreak from 2007-2010 traced to dairy goats resulted in over 4,100 human cases with estimated costs of more than 300 million euros. Smaller human Q fever outbreaks of small ruminant origin have occurred in the United States, and characterizing shedding is important to understand the risk of future outbreaks. In this study, we assessed bacterial shedding and seroprevalence in 100 sheep from an Idaho location associated with a 1984 human Q fever outbreak. We observed 5% seropositivity, which was not significantly different from the national average of 2.7% for the U.S. (P>0.05). Furthermore, C. burnetii was not detected by quantitative PCR from placentas, vaginal swabs, or fecal samples. Specifically, a three-target quantitative PCR of placenta identified 0.0% shedding (exact 95% confidence interval: 0.0%-2.9%). While presence of seropositive individuals demonstrates some historical C. burnetii exposure, the placental sample confidence interval suggests 2016 shedding events were rare or absent. The location maintained the flock with little or no depopulation in 1984 and without C. burnetii vaccination during or since 1984. It is not clear how a zero-shedding rate was achieved in these sheep beyond natural immunity, and more work is required to discover and assess possible factors that may contribute towards achieving zero-shedding status. We provide the first U.S. sheep placental C. burnetii shedding update in over 60 years and demonstrate potential for C. burnetii shedding to reach undetectable levels after an outbreak event even in the absence of targeted interventions, such as vaccination.

Eleven laboratories collaborated to determine the periodic prevalence of Salmonella in a population of dogs and cats in the United States visiting veterinary clinics. Fecal samples (2,965) solicited from 11 geographically dispersed veterinary testing laboratories were collected in 36 states between January 2012 and April 2014 and tested using a harmonized method. The overall study prevalence of Salmonella in cats (3 of 542) was Salmonella -positive dogs were significantly more likely to have consumed raw food ( P = 0.01), to have consumed probiotics ( P = 0.002), or to have been given antibiotics ( P = 0.01). Rural dogs were also more likely to be Salmonella positive than urban ( P = 0.002) or suburban ( P = 0.001) dogs. In the 67 isolates, 27 unique serovars were identified, with three dogs having two serovars present. Antimicrobial susceptibility testing of 66 isolates revealed that only four of the isolates were resistant to one or more antibiotics. Additional characterization of the 66 isolates was done using pulsed-field gel electrophoresis and whole-genome sequencing (WGS). Sequence data compared well to resistance phenotypic data and were submitted to the National Center for Biotechnology Information (NCBI). This study suggests an overall decline in prevalence of Salmonella -positive dogs and cats over the last decades and identifies consumption of raw food as a major risk factor for Salmonella infection. Of note is that almost half of the Salmonella -positive animals were clinically nondiarrheic.

Background Salmonella enterica can significantly impact management of animal facilities. Comprehensive screening is essential for effective control in high-risk populations. Availability of reliable point-of-care diagnostic tests would facilitate these efforts. Hypothesis/Objectives Compare the ability of commercially available rapid diagnostic assays (2 lateral flow immunoassays [LFIs], DNA hybridization [DNAH], real-time PCR [qPCR]), and culture to detect common serotypes of S. enterica in feces. Animals n/a. Methods In an experimental study, 112 S. enterica isolates were randomly selected from the 10 most common serotypes recovered at a veterinary hospital. Archived isolates were amplified in broth and standardized inocula (100 colony forming units) were incubated with equine feces in tetrathionate broth (TET). Cultures were tested in a blinded fashion by using LFIs, DNAH, qPCR, and culture. Results The LFIs detected 84% and 67% of isolates, respectively, but reactivity varied among serotypes. Both reacted poorly with serotype Cerro (Group K) isolates, and 1 LFI did not react with any serotype Mbandaka (Group C1) or Montevideo (Group C1) isolates. DNAH detected 94% of isolates, whereas culture and qPCR most reliably detected all serotypes. False-positive results were obtained for 4 negative controls by using DNAH and 1 negative control by using qPCR, but LFIs and culture had no false-positive results. Conclusions and Clinical Importance Culture, qPCR, and DNAH were effective in detecting most Salmonella isolates, but have limited application at point-of-care settings. LFIs are appealing as point-of-care tests because of low cost and ease of use, but limited detection of some serotypes needs to be evaluated with samples obtained from naturally infected animals.

Wild birds are the primary source of genetic diversity for influenza A viruses that eventually emerge in poultry and humans. Much progress has been made in the descriptive ecology of avian influenza viruses (AIVs), but contributions are less evident from quantitative studies (e.g., those including disease dynamic models). Transmission between host species, individuals and flocks has not been measured with sufficient accuracy to allow robust quantitative evaluation of alternate control protocols. We focused on the United States of America (USA) as a case study for determining the state of our quantitative knowledge of potential AIV emergence processes from wild hosts to poultry. We identified priorities for quantitative research that would build on existing tools for responding to AIV in poultry and concluded that the following knowledge gaps can be addressed with current empirical data: (1) quantification of the spatio-temporal relationships between AIV prevalence in wild hosts and poultry populations, (2) understanding how the structure of different poultry sectors impacts within-flock transmission, (3) determining mechanisms and rates of between-farm spread, and (4) validating current policy-decision tools with data. The modeling studies we recommend will improve our mechanistic understanding of potential AIV transmission patterns in USA poultry, leading to improved measures of accuracy and reduced uncertainty when evaluating alternative control strategies.

Summary Salmonella enterica is a common zoonotic pathogen in humans. Transmission typically occurs through consumption of contaminated food products or contact with infected animals, including poultry or their environment. The objective of this study was to estimate the frequency of Salmonella contamination in the environment in poultry exhibits at agricultural fairs. Samples were collected from cages, feed, floors and tables in the exhibit and cultured for Salmonella. At least one environmental sample was positive for Salmonella in 10 of 11 fairs (91%), and Salmonella was isolated from 28 of 55 environmental samples (50.9%). Eleven different serotypes were detected. Results of this study demonstrate that environmental surfaces at agricultural fairs can be contaminated with Salmonella and could potentially serve as a route of transmission to bird owners and the general public. Poultry owners and the general public should be educated about the risks of Salmonella infection from the poultry exhibit environment. Agricultural fairs should consider instituting policies and practices to improve hygiene and mitigate the risk of zoonotic salmonellosis.

Coxiella burnetii, a zoonotic bacterium, has recently been identified in several marine mammal species on the Pacific Coast of North America, but little is known about the epidemiology, transmission, and pathogenesis in these species. We tested sera archived from northern fur seals (NFS, Callorhinus ursinus; n=236) and Steller sea lions (SSL, Eumetopias jubatus; n=72) sampled in Alaska for C. burnetii antibodies, and vaginal swabs from NFS (n=40) for C. burnetii by qPCR. The antibody prevalence in NFS samples from 2009 and 2011 (69%) was significantly higher than in 1994 (49%). The antibody prevalence of SSL samples from 2007 to 2011 was 59%. All NFS vaginal swabs were negative for C. burnetii, despite an 80% antibody prevalence in the matched sera. The significant increase in antibody prevalence in NFS from 1994 to 2011 suggests that the pathogen may be increasingly common or that there is marked temporal variation within the vulnerable NFS population. The high antibody prevalence in SSL suggests that this pathogen may also be significant in the endangered SSL population. These results confirm that C. burnetii is more prevalent within these populations than previously known. More research is needed to determine how this bacterium may affect individual, population, and reproductive health of marine mammals.

Since 1997, highly pathogenic avian influenza subtype H5N1 has emerged to cause severe disease in humans in over 15 countries. Humans who have regular contact with poultry or wild birds may be at greater risk of infection with highly pathogenic avian influenza and other zoonotic avian diseases. To develop preventative measures for transmission of avian influenza to high-risk human populations in the United States, we examined human-bird interactions in the upland gamebird industry. Upland gamebird permit holders were surveyed for information on human-bird contact, biosecurity practices, facility management practices, flock/release environment, and bird health. Results suggest that some upland gamebird facilities provide an environment for extensive and intimate human-bird interaction such that humans associated with these facilities may be at greater risk for zoonotic disease transmission.

Raccoons can transmit avian and human influenza Influenza Infection in Wild Raccoons, Raccoons (Procyon lotor) are common, widely distributed animals that frequently come into contact with wild waterfowl, agricultural operations, and humans. Serosurveys showed that raccoons are exposed to avian influenza virus. We found antibodies to a variety of influenza virus subtypes (H10N7, H4N6, H4N2, H3, and H1) with wide geographic variation in seroprevalence. Experimental infection studies showed that raccoons become infected with avian and human influenza A viruses, shed and transmit virus to virus-free animals, and seroconvert. Analyses of cellular receptors showed that raccoons have avian and human type receptors with a similar distribution as found in human respiratory tracts. The potential exists for co-infection of multiple subtypes of influenza virus with genetic reassortment and creation of novel strains of influenza virus. Experimental and field data indicate that raccoons may play an important role in influenza disease ecology and pose risks to agriculture and human health.

Swine play an important role in the disease ecology of influenza. Having cellular receptors in common with birds and humans, swine provide opportunities for mixed infections and potential for genetic reassortment between avian, human, and porcine influenza. Feral swine populations are rapidly expanding in both numbers and range and are increasingly coming into contact with waterfowl, humans, and agricultural operations. In this study, over 875 feral swine were sampled from six states across the United States for serologic evidence of exposure to influenza. In Oklahoma, Florida, and Missouri, USA, no seropositive feral swine were detected. Seropositive swine were detected in California, Mississippi, and Texas, USA. Antibody prevalences in these states were 1% in Mississippi, 5% in California, and 14.4% in Texas. All seropositive swine were exposed to H3N2 subtype, the predominant subtype currently circulating in domestic swine. The only exceptions were in San Saba County, Texas, where of the 15 seropositive samples, four were positive for H1N1 and seven for both H1N1 and H3N2. In Texas, there was large geographical and temporal variation in antibody prevalence and no obvious connection to domestic swine operations. No evidence of exposure to avian influenza in feral swine was uncovered. From these results it is apparent that influenza in feral swine poses a risk primarily to swine production operations. However, because feral swine share habitat with waterfowl, prey on and scavenge dead and dying birds, are highly mobile, and are increasingly coming into contact with humans, the potential for these animals to become infected with avian or human influenza in addition to swine influenza is a distinct possibility.

Influenza A viruses pose significant health and economic threats to humans and animals. Outbreaks of avian influenza virus (AIV) are a liability to the poultry industry and increase the risk for transmission to humans. There are limitations to using the AIV vaccine in poultry, creating barriers to controlling outbreaks and a need for alternative effective control measures. Application of RNA interference (RNAi) techniques hold potential; however, the delivery of RNAi-mediating agents is a well-known obstacle to harnessing its clinical application. We introduce a novel antiviral approach using bacterial vectors that target avian mucosal epithelial cells and deliver (small interfering RNA) siRNAs against two AIV genes, nucleoprotein (NP) and polymerase acidic protein (PA). Using a red fluorescent reporter, we first demonstrated vector delivery and intracellular expression in avian epithelial cells. Subsequently, we demonstrated significant reductions in AIV shedding when applying these anti-AIV vectors prophylactically. These antiviral vectors provided up to a 10,000-fold reduction in viral titers shed, demonstrating in vitro proof-of-concept for using these novel anti-AIV vectors to inhibit AIV shedding. Our results indicate this siRNA vector technology could represent a scalable and clinically applicable antiviral technology for avian and human influenza and a prototype for RNAi-based vectors against other viruses.

Valvular endocarditis has been well described in northern sea otters Enhydra lutris kenyoni of Alaska and in many cases no cause has been identified. It is also one of the most com- mon conditions observed in people with chronic Coxiella burnetii infection. Given the high levels of C. burnetii exposure in marine mammals distributed throughout the same geographic range as the northern sea otter, and the presence of valvular lesions seen in otters, the objective of this study was to determine the level of C. burnetii exposure in otters and investigate any association between exposure, infection and valvular disease in this species. Archived serum from 75 live cap- tured, apparently healthy otters (25 from each of 3 stocks) and 30 dead otters were tested for C. burnetii antibodies by indirect florescent antibody assay (IFA). Archived bone marrow and heart valves were tested for C. burnetii DNA by real-time PCR (qPCR). Overall, the seroprevalence in live otters was 17%, with significantly more exposed animals in the south central (40%) stock rel- ative to the southwest (8%) and southeast (4%). The seroprevalence of animals sampled post mortem was 27%, although none of the bone marrow or heart valve samples were positive by qPCR. Results of this study failed to demonstrate a significant association between C. burnetii infection and valvular endocarditis in sea otters; however, the differing seroprevalence suggests that exposure opportunities vary geographically.

The Waved Albatross (Phoebastria irrorata) is a critically endangered seabird in a rapidly shrinking population in the Galápagos Islands, Ecuador. The introduction of novel pathogens and parasites poses a threat to population persistence. Monitoring disease prevalence and guarding against the spread of such agents in endemic taxa are conservation priorities for the Galápagos, where recent increases in the prevalence of avian pox may have contributed to population declines and range contractions in other bird species. During November 2013-January 2014, we identified 14 Waved Albatross nestlings at our study site on Española Island with avian pox-like lesions and clinical signs. Other seabirds, landbirds, and adult Waved Albatrosses were apparently unaffected. Histopathology of tissue samples from five infected nestlings revealed inclusion bodies in all samples, consistent with avipoxvirus infection. We documented higher mortality (6 of 14 nestlings) in affected nestlings than in unaffected young in this small outbreak of avian pox, the first report of its kind in the world's only tropical albatross.

SUMMARYAs feral swine continue to expand their geographical range and distribution across the United States, their involvement in crop damage, livestock predation, and pathogen transmission is likely to increase. Despite the relatively recent discovery of feral swine involvement in the aetiology of a variety of pathogens, their propensity to transmit and carry a wide variety of pathogens is disconcerting. We examined sera from 2055 feral swine for antibody presence to six serovars of Leptospira that can also infect humans, livestock or domestic animals. About 13% of all samples tested positive for at least one serovar, suggesting that Leptospira infection is common in feral swine. Further studies to identify the proportion of actively infected animals are needed to more fully understand the risk they pose.

The Southwest United States, including Arizona and New Mexico, has a diverse climate and is home to many different avian species. We sequenced the hemagglutinin (HA) gene of twenty influenza specimens for the years 2007-2009. This included four from Arizona, and sixteen from New Mexico. We analyzed the sequences and determined the following HA subtypes: H3, H4, H6, H8, and H11. For each subtype, we combined our virus sequences with those from a public database, and inferred phylogeographic models of influenza diffusion. Statistical phylogeography indicated that overall evolutionary diffusion of avian influenza viruses is geographically structured (p

Influenza A virus subtype H1N1 A(H1N1)pdm09 was first confirmed in pigs in the United States in October 2009. In November 2010, lungs and intestines from 2 York piglets from a small, privately owned herd were submitted to the Colorado State University Veterinary Diagnostic Laboratory. The submitting veterinarian reported rapid weight loss and signs of pneumonia in the piglets. Gross lesions included caudoventral pneumonia in both piglets, and histologic lesions in the lungs showed characteristics consistent with influenza virus and bacterial infection. Ribonucleic acid extracted from fresh lung homogenates from both piglets was positive for influenza A(H1N1)pdm09 by a real-time reverse transcription polymerase chain reaction. Virus was isolated from lung homogenates from both piglets in Madin–Darby canine kidney cells, as well as in 10-day-old specific pathogen–free embryonated chicken eggs. Sequence analysis showed 98% homology with 2009 H1N1 human isolates from across the United States and 98% homology against two 2009 and 2010 swine isolates from Nebraska and Minnesota. The current report documents the possible transmission of pandemic influenza A(H1N1)2009 virus [A(H1N1)pdm09] from a human being to a small, privately owned backyard swine herd. The owner was employed as a pharmacist, making occupational exposure to the pandemic influenza A(H1N1)pdm09 a possibility.

Background: The northern fur seal (Callorhinus ursinus) is an important cultural and nutritional resource for the Aleut community on St. Paul Island Alaska. In recent years, an increasing number of zoonotic pathogens have been identified in the population, but the public health significance of these findings is unknown. To determine the prevalence of Coxiella burnetii and Brucella spp. in northern fur seal tissues, eight tissue types from 50 subsistence-harvested fur seals were tested for bacterial DNA by real-time polymerase chain reaction. Findings: Of the 400 samples tested, only a single splenic sample was positive for Brucella spp. and the cycle threshold (ct) value was extremely high suggesting a low concentration of DNA within the tissue. C. burnetii DNA was not detected. Conclusions: Findings suggest that the risk of humans contracting brucellosis or Q fever from the consumption of harvested northern fur seals is low.