ABSTRACT Pneumonia is a pervasive, population‐limiting disease of bighorn sheep (Ovis canadensis) with limited options for management. We conducted a selective removal experiment in two regions (north and south) of the Hells Canyon bighorn sheep metapopulation to test the hypothesis that pneumonia is maintained in bighorn sheep populations by chronic carriers of the bacterium Mycoplasma ovipneumoniae. We detected M. ovipneumoniae in 83 adults over 11 years across seven study populations. We removed five carriers of M. ovipneumoniae and nine non‐carriers from two treatment populations in northern Hells Canyon and 15 chronic carriers from a treatment population in southern Hells Canyon. We did not remove any sheep from four control populations. Local elimination of M. ovipneumoniae in the two northern treatment populations within a year after removals was indicated by no further detection of the pathogen, waning antibody levels, and lack of antibody in animals born after removals. Elimination in treatment populations was followed by fadeout of M. ovipneumoniae in the four adjacent control populations over the next 4 years without any further removals. Selective removals were associated with a decline in prevalence but did not eliminate M. ovipneumoniae in the southern treatment population. Clearance of infection led to nearly doubling of survival over the first 4 months of life, a 74% increase in recruitment to 7–10 months of age, and an increase in the average annual rate of population growth from 1% to 12%. The results of this experiment provide support for a focus on carriers of M. ovipneumoniae for mitigating low lamb recruitment associated with pneumonia‐induced mortality observed in many bighorn sheep populations across North America. However, mixed outcomes indicate that a better understanding of infection persistence and fadeout could increase the effectiveness of management interventions.

ABSTRACT We hypothesized that bighorn sheep ewes with chronic nasal Mycoplasma ovipneumoniae carriage are the source of infection that results in fatal lamb pneumonia. We tested this hypothesis in captive bighorn ewes at two study facilities over a 5-year period, by identifying carrier ewes and then comparing lamb fates in groups that did (exposed pens) or did not (non-exposed pens) include one or more carrier ewes. Most (23 of 30) lambs born in exposed pens, but none of 11 lambs born in non-exposed pens, contracted fatal pneumonia. In addition, surviving lambs in exposed pens showed obvious signs of respiratory disease while lambs in non-exposed pens did not. In crossover experiments, individual non-carrier ewes had lambs that experienced fatal pneumonia in years when housed in exposed pens, but not in years when housed in non-exposed pens. The results of these studies clearly associate lamb pneumonia to exposure to M. ovipneumoniae carrier ewes, consistent with a necessary role for this agent in epizootic pneumonia of bighorn sheep. These data specifically highlight the role of chronic M. ovipneumoniae carriage by some bighorn ewes in the epidemiology of this population-limiting wildlife disease. IMPORTANCE Bighorn sheep populations, historically important in mountain and canyon ecosystems of western North America, declined precipitously following European settlement of North America and remain depressed today. One factor contributing to these declines and lack of recovery is epizootic pneumonia caused by the bacterium Mycoplasma ovipneumoniae . This pathogen arrived with settlers’ domestic sheep and goats and spilled over to infect bighorn sheep, a process that continues to this day. Bighorn losses from this disease include high rates of mortality (median, approaching 50%) of all ages of bighorn sheep on initial exposure, followed in subsequent years to decades by mortality largely limited to young lambs. The source of infection causing persistent lamb losses is the focus of the research described here. Conducting these studies on groups of captive bighorn sheep enabled demonstration of clear linkage between largely asymptomatic nasal carriage of M. ovipneumoniae by ewes and outbreaks of fatal pneumonia in lambs.

Mycoplasma ovipneumoniae is associated with respiratory disease in wild and domestic Caprinae globally, with wide variation in disease outcomes within and between host species. To gain insight into phylogenetic structure and mechanisms of pathogenicity for this bacterial species, we compared M. ovipneumoniae genomes for 99 samples from 6 countries (Australia, Bosnia and Herzegovina, Brazil, China, France and USA) and 4 host species (domestic sheep, domestic goats, bighorn sheep and caribou). Core genome sequences of M. ovipneumoniae assemblies from domestic sheep and goats fell into two well-supported phylogenetic clades that are divergent enough to be considered different bacterial species, consistent with each of these two clades having an evolutionary origin in separate host species. Genome assemblies from bighorn sheep and caribou also fell within these two clades, indicating multiple spillover events, most commonly from domestic sheep. Pangenome analysis indicated a high percentage (91.4 %) of accessory genes (i.e. genes found only in a subset of assemblies) compared to core genes (i.e. genes found in all assemblies), potentially indicating a propensity for this pathogen to adapt to within-host conditions. In addition, many genes related to carbon metabolism, which is a virulence factor for Mycoplasmas, showed evidence for homologous recombination, a potential signature of adaptation. The presence or absence of annotated genes was very similar between sheep and goat clades, with only two annotated genes significantly clade-associated. However, three M. ovipneumoniae genome assemblies from asymptomatic caribou in Alaska formed a highly divergent subclade within the sheep clade that lacked 23 annotated genes compared to other assemblies, and many of these genes had functions related to carbon metabolism. Overall, our results suggest that adaptation of M. ovipneumoniae has involved evolution of carbon metabolism pathways and virulence mechanisms related to those pathways. The genes involved in these pathways, along with other genes identified as potentially involved in virulence in this study, are potential targets for future investigation into a possible genomic basis for the high variation observed in disease outcomes within and between wild and domestic host species.

Escherichia coli is the most common cause of recurrent urinary tract infection (UTI) in dogs. UTI recurrence comprises of persistent, unresolved E. coli infection or reinfection with a different strain of E. coli. Differentiating between these processes is clinically important but is often impossible with routine diagnostics. We tested the hypothesis that most recurrent canine E. coli bacteriuria is due to recurrence of the same E. coli strain involved in the initial infection. Molecular typing was performed on 98 urinary E. coli isolated from dogs with recurrent bacteriuria from five veterinary diagnostic laboratories in the United States. Of the 42 dogs in this study with multiple E. coli bacteriuria observations, a single strain of E. coli caused recurrent bacteriuria in 26 (62 %) dogs, in some cases on multiple occasions for prolonged periods of time (up to eight months). A single E. coli strain was detected during both subclinical bacteriuria and clinically-apparent UTI in three dogs. Isolates with the P-fimbrial adhesin genes papA and papC were associated with recurrence by the same strain of E. coli. Multiple isolations of a single strain of E. coli associated with recurrent bacteriuria suggests that E. coli may be maintained within the urinary tract of some dogs for prolonged periods of time. In some patients, the same strain can cause both clinical UTI and subclinical bacteriuria. This indicates that in dogs, the urinary bladder may serve as a subclinical, long-term reservoir of E. coli that may cause clinical UTI in the future.

Introduction Throughout their range, bighorn sheep ( Ovis canadensis ) populations have seen significant disease-associated declines. Unfortunately, understanding of the underlying epidemiological processes driving the disease dynamics in this species has hindered conservation efforts aimed at improving the health and long-term viability of these populations. Individual response to pathogen exposure emerges from dynamic interactions between competing evolutionary processes within the host and pathogen. The host’s adaptive immune system recognizes pathogens and mounts a defensive response. Pathogens have evolved strategies to overcome adaptive immune defenses including maintaining high genetic diversity through rapid evolution. The outcomes of this evolutionary warfare determine the success of pathogen invasion of the host and ultimately the success of conservation efforts. Methods During an epizootic dominated by a single strain, we explore these host-pathogen dynamics by examining the variation in effects of pathogen invasion on captive bighorn sheep with differing histories of exposure to genetically diverse strains of Mycoplasma ovipneumoniae (Movi). We monitored clinical signs of disease and sampled animals and their environment to detect spread of Movi among 37 bighorn sheep separated into nine pens based on known exposure histories. Results We documented Movi transmission within and across pens and we detected Movi DNA in air, water, and invertebrate samples. Higher levels of antibody to Movi prior to the epizootic were associated with a lower likelihood of presenting clinical signs of pneumonia. Nonetheless, higher antibody levels in symptomatic individuals were associated with more severe progressive disease, increased probability and speed of pneumonia-induced mortality, and reduced likelihood of returning to a healthy state. Bighorn sheep with previous exposure to a strain other than the predominant epizootic strain were more likely to recover. Discussion Our results indicate that Movi-strain variability was sufficient to overwhelm the adaptive host immunological defenses. This outcome indicates, in free-ranging herds, past exposure is likely insufficient to protect bighorn sheep from infection by new Movi strains, although it influences the progression of disease and recovery within the herd. Therefore, given Movi-strain variability and the lack of immunological protection from past exposure, focusing management efforts on minimizing the introduction of Movi into bighorn herds, through separation of domestic and bighorn sheep and avoidance of management activities that create commingling of bighorn sheep carrying differing Movi strains, will likely be the most effective approach for reducing the effects of disease and achieving bighorn sheep conservation goals.

Mycoplasma ovipneumoniae (M. ovipneumoniae) is a respiratory pathogen associated with mild to moderate respiratory disease in domestic lambs and severe pneumonia outbreaks in wild ruminants such as bighorn sheep. However, whether M. ovipneumoniae by itself causes clinical respiratory disease in domestic sheep in the absence of secondary bacterial pathogens is still unclear. The goal of our study was to better understand the role of M. ovipneumoniae as a respiratory pathogen in domestic sheep and to explore potential antibiotic treatment approaches. Therefore, we inoculated four 4-month-old, specific-pathogen-free lambs with fresh nasal wash fluids from M. ovipneumoniae-infected sheep. The lambs were monitored for M. ovipneumoniae colonization, M. ovipneumoniae-specific antibodies, clinical signs, and cellular and molecular correlates of lung inflammation for eight weeks. All lambs then were treated with gamithromycin and observed for an additional four weeks. M. ovipneumoniae inoculation resulted in stable colonization of the upper respiratory tract in all M. ovipneumoniae-inoculated, but in none of the four mock-infected control lambs. All M. ovipneumoniae-infected lambs developed a robust antibody response to M. ovipneumoniae within 2 weeks. However, we did not observe significant signs of respiratory disease, evidence of lung damage or inflammation in any of the infected lambs. Interestingly, treatment with gamithromycin, which blocked growth of the M. ovipneumoniae in vitro, failed to reduce M. ovipneumoniae colonization. These observations indicate that, in the absence of co-infections, M. ovipneumoniae caused asymptomatic colonization of the upper respiratory tract that was resistant to clearance by the host immune response and by gamithromycin treatment.

A 2013 outbreak of respiratory disease in bighorn sheep from California's Mojave Desert metapopulation caused high mortality in at least one population. Subsequent PCR and strain-typing indicate widespread infection of a single strain of Mycoplasma ovipneumoniae throughout this region. Serosurvey of archived samples showed that some populations have had antibodies to M. ovipneumoniae since at least 1986, although pre-2013 strain-type data are unavailable.

The bacterium E. coli O157:H7 causes foodborne disease in humans that can lead to bloody diarrhea, kidney failure, vascular damage, and death. Healthy cattle are the main source of this human pathogen. Reducing E. coli O157:H7 in cattle will reduce human disease. Using a randomized comparison, a bovine vaccine to reduce carriage of the human pathogen was tested. A detoxified E. coli O157:H7 strain, missing genes that cause disease, was fed to cattle as an oral vaccine to reduce carriage of pathogenic E. coli O157:H7. After vaccination, the cattle were challenged with disease-causing E. coli O157:H7. The vaccinated cattle had decreased E. coli O157:H7 during the first 7 days postchallenge and shed the bacteria for a shorter duration than the nonvaccinated control cattle. The results support optimization of the approach to cattle vaccination that would reduce human disease.

Abstract A respiratory disease epizootic at the National Bison Range (NBR) in Montana in 2016–2017 caused an 85% decline in the bighorn sheep population, documented by observations of its unmarked but individually identifiable members, the subjects of an ongoing long‐term study. The index case was likely one of a small group of young bighorn sheep on a short‐term exploratory foray in early summer of 2016. Disease subsequently spread through the population, with peak mortality in September and October and continuing signs of respiratory disease and sporadic mortality of all age classes through early July 2017. Body condition scores and clinical signs suggested that the disease affected ewe groups before rams, although by the end of the epizootic, ram mortality (90% of 71) exceeded ewe mortality (79% of 84). Microbiological sampling 10 years to 3 months prior to the epizootic had documented no evidence of infection or exposure to Mycoplasma ovipneumoniae at NBR, but during the epizootic, a single genetic strain of M. ovipneumoniae was detected in affected animals. Retrospective screening of domestic sheep flocks near the NBR identified the same genetic strain in one flock, presumptively the source of the epizootic infection. Evidence of fatal lamb pneumonia was observed during the first two lambing seasons following the epizootic but was absent during the third season following the death of the last identified M. ovipneumoniae carrier ewe. Monitoring of life‐history traits prior to the epizootic provided no evidence that environmentally and/or demographically induced nutritional or other stress contributed to the epizootic. Furthermore, the epizootic occurred despite proactive management actions undertaken to reduce risk of disease and increase resilience in this population. This closely observed bighorn sheep epizootic uniquely illustrates the natural history of the disease including the (presumptive) source of spillover, course, severity, and eventual pathogen clearance.

Bighorn sheep (Ovis canadensis) were once extirpated from the Black Hills region of South Dakota, U.S.A., mirroring declining populations throughout North America. Since the 1960s, several reintroductions have occurred in the Black Hills to reestablish populations, with varying success. We translocated 26 bighorn sheep from Alberta, Canada, to the Black Hills (February 2015) to restore bighorn sheep to their historic range. Due to prior examinations of cause‐specific survival, subsequent genetic diversity and disease prevalence analyses were required to evaluate success of the restoration effort. We measured a mean allelic diversity of 5.23 (SE = 0.44 [mean number of alleles]) and an observed heterozygosity of 0.71 (SE = 0.06; expected = 0.64 ± 0.05) in the translocated individuals. Translocated bighorn sheep tested negative for Mycoplasma ovipneumoniae at capture. An autogenous vaccine was administered prior to release in an attempt to safeguard the translocated bighorn sheep from infection with a strain known to be resident in adjacent bighorn sheep populations. However, the year following the translocation, a different strain of M. ovipneumoniae was associated with a pneumonia outbreak that resulted in 57.9% mortality. Our results suggest that allelic diversity and heterozygosity were sufficient for long‐term herd establishment, reducing the potential for founder effects. However, the overwhelming mortality associated with pneumonia, via the transfer of M. ovipneumoniae from an unknown source, limited the success or our reintroduction efforts. Successful attempts to restore bighorn sheep to their historic ranges must consider and mitigate potential routes for M. ovipneumoniae transmission pre‐ and post‐reintroduction.

For most pathogens, testing procedures can be used to distinguish between different strains with which individuals are infected. Due to the growing availability of such data, multistrain models have increased in popularity over the past few years. Quantifying the interactions between different strains of a pathogen is crucial in order to obtain a more complete understanding of the transmission process, but statistical methods for this type of problem are still in the early stages of development. Motivated by this demand, we construct a stochastic epidemic model that incorporates additional strain information and propose a statistical algorithm for efficient inference. The model improves upon existing methods in the sense that it allows for both imperfect diagnostic test sensitivities and strain misclassification. Extensive simulation studies were conducted in order to assess the performance of our method, while the utility of the developed methodology is demonstrated on data obtained from a longitudinal study of Escherichia coli O157:H7 strains in feedlot cattle.

Abstract Chronic pathogen carriage is one mechanism that allows diseases to persist in populations. We hypothesized that persistent or recurrent pneumonia in bighorn sheep (Ovis canadensis) populations may be caused by chronic carriers of Mycoplasma ovipneumoniae (Mo). Our experimental approach allowed us to address a conservation need while investigating the role of chronic carriage in disease persistence. We tested our hypothesis in two bighorn sheep populations in South Dakota, USA. We identified and removed Mo chronic carriers from the Custer State Park (treatment) population. Simultaneously, we identified carriers but did not remove them from the Rapid City population (control). We predicted removal would result in decreased pneumonia, mortality, and Mo prevalence. Both population ranges had similar habitat and predator communities but were sufficiently isolated to preclude intermixing. We classified chronic carriers as adults that consistently tested positive for Mo carriage over a 20‐month sampling period (n = 2 in the treatment population; n = 2 in control population). We failed to detect Mo or pneumonia in the treatment population after chronic carrier removal, while both remained in the control. Mortality hazard for lambs was reduced by 72% in the treatment population relative to the control (CI = 36%, 91%). There was also a 41% reduction in adult mortality hazard attributable to the treatment, although this was not statistically significant (CI = 82% reduction, 34% increase). Synthesis and Applications: These results support the hypothesis that Mo is a primary causative agent of persistent or recurrent respiratory disease in bighorn sheep populations and can be maintained by a few chronic carriers. Our findings provide direction for future research and management actions aimed at controlling pneumonia in wild sheep and may apply to other diseases.

Our objective was to determine whether beta-lactamase genes are carried within bacteriophage capsids, as a first step towards exploring the possible role of bacteriophages as vehicles for dispersal of antimicrobial resistance genes through an agricultural region of Washington State. Water samples (n = 178) from municipal wastewater treatment plants, river and irrigation canals were collected over a period of eight months. The occurrence of four beta-lactam resistance gene groups (bla(TEM), bla(CTX-M), bla(PSE) and bla(CMY-2)) and three carbapenem resistance genes (bla(KPC), bla(OXA-48)-like, and bia(NDM)) in bacterial and phage fractions of water samples was evaluated by PCR. All of the seven targeted resistance genes were detected both in wastewater and river water samples. Relatively high proportions of samples (7.3%-64.9%) positive for resistance genes were found in bacteriophage fractions of water samples compared to the bacterial fractions (5.4%-36.8%). bla(OXA-48)-like (57.3%) and bla(TEM) (64.0%) were the most prevalent antimicrobial resistance genes detected at all the sampling points. Resistance genes are commonly present in treated wastewater flowing through municipal and agricultural environments, indicating a plausible role for this water in the dissemination of antimicrobial resistance traits, including bla(CTX-M). (C) 2019 Elsevier Ltd. All rights reserved.

Farmland biodiversity benefits pollination, biological control and other key ecosystem services. Food safety has been seen as an exception to this broader pattern, as diverse farmlands attract wildlife that vector foodborne human pathogens. Resulting mitigation efforts thus often seek to deter wildlife by removing natural habitats, while also excluding vertebrate livestock. However, surprising recent evidence suggests that farm simplification actually increases the likelihood that produce will be contaminated with human pathogens. Here, we consider the possibility that intensified agriculture harms faeces-feeding (coprophagous) beetles and bacteria, which could contribute to heightened food-safety risk. In 70 commercial vegetable fields spanning the US west coast, using either organic or conventional farming methods, we surveyed coprophages both above- and below-ground. We also measured removal rates of the faeces of Sus scrofa, which vectors foodborne pathogens both as livestock and as feral wildlife. Above-ground, organic farms fostered dung beetle species that removed S.scrofa faeces more rapidly than was seen on conventional farms, although this benefit was weakened in simplified landscapes dominated by pasture and an introduced dung beetle. Below-ground, organic farming encouraged significantly higher biodiversity among soil bacteria. Organic farming similarly benefitted dung beetles and bacteria on farms that produced livestock alongside vegetables, or vegetables alone. Complementary laboratory experiments revealed that the dung beetle species and biodiverse bacterial assemblages typical of organic farms were significantly more effective at suppressing human-pathogenic Escherichia coli O157:H7, compared to coprophage communities associated with conventional farms. This suggests that farm management practices, coprophage conservation, and human-pathogen suppression might be linked.Synthesis and applications. Altogether, our results indicate that insects and microbes can rapidly remove faeces, with the potential to also decrease the persistence of human pathogens. In turn, this suggests that improved food safety may be an important, and perhaps underappreciated, ecosystem service that is enhanced by on-farm biodiversity. We recommend that farm managers and regulators consider the risks and benefits to coprophages when making management decisions regarding food safety. Altogether, our results indicate that insects and microbes can rapidly remove faeces, with the potential to also decrease the persistence of human pathogens. In turn, this suggests that improved food safety may be an important, and perhaps underappreciated, ecosystem service that is enhanced by on-farm biodiversity. We recommend that farm managers and regulators consider the risks and benefits to coprophages when making management decisions regarding food safety. Editor's Choice

Association of Mycoplasma ovipneumoniae with pneumonia in domestic small ruminants has been described in Europe, Asia, and New Zealand but has received less attention in the United States. In 2011, the US Department of Agriculture's National Animal Health Monitoring System detected M. ovipneumoniae shedding in 88% of 453 domestic sheep operations tested in 22 states that accounted for 85.5% of US ewe inventory in 2001. We evaluated factors associated with M. ovipneumoniae infection presence and prevalence, and we compared health, lamb production, and ewe losses in infected and uninfected operations. M. ovipneumoniae detection was more common in larger operations than in smaller operations. Both likelihood of detection (at the operation level) and within-operation prevalence were higher in operations with more open management practices than in operations with more closed management practices. M. ovipneumoniae-positive operations showed significantly lower lambing rates and lower rates of lamb survival to weaning after accounting for differences in operation size and management practice. While its effect on any single rate was not particularly large, in aggregate we estimated that M. ovipneumoniae presence was associated with an approximately 4.3% reduction in annual lamb production.

Spillover diseases have significant consequences for human and animal health, as well as wildlife conservation. We examined spillover and transmission of the pneumonia-associated bacterium Mycoplasma ovipneumoniae in domestic sheep, domestic goats, bighorn sheep, and mountain goats across the western United States using 594 isolates, collected from 1984 to 2017. Our results indicate high genetic diversity of M. ovipneumoniae strains within domestic sheep, whereas only one or a few strains tend to circulate in most populations of bighorn sheep or mountain goats. These data suggest domestic sheep are a reservoir, while the few spillovers to bighorn sheep and mountain goats can persist for extended periods. Domestic goat strains form a distinct clade from those in domestic sheep, and strains from both clades are found in bighorn sheep. The genetic structure of domestic sheep strains could not be explained by geography, whereas some strains are spatially clustered and shared among proximate bighorn sheep populations, supporting pathogen establishment and spread following spillover. These data suggest that the ability to predict M. ovipneumoniae spillover into wildlife populations may remain a challenge given the high strain diversity in domestic sheep and need for more comprehensive pathogen surveillance.

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Some of the greatest successes in infectious disease control rest on empirically grounded models of human and livestock infections. In contrast, disease control in wildlife has not always been as successful. Timely translation of knowledge into proposed management actions remains a challenge in several wildlife disease systems, one of which is pneumonia management in bighorn sheep throughout the North American West. Although pneumonia was recognised as a major impediment to bighorn sheep conservation >80 years ago, a series of challenges stymied the management decision-making process. Despite past obstacles, recent advances from long-term, intensive studies of marked individual sheep have motivated new interest in research-driven strategies for disease management in this system. The system provides an unusual opportunity to study an emerging pathogen disproportionately impacting immature animals through infections that originate from asymptomatically infected adult hosts. We tell the story of bighorn sheep pneumonia, emphasising the obstacles that historically hindered decision-making, the biological or logistical constraints underlying each decision point, and the particular empirical insights that clarified each constraint.

•M. ovipneumoniae quantitation by qPCR and flow cytometry were closely correlated with CCU50.•CCU50 and qPCR demonstrated wide ranges of detection.•Flow cytometry consistently underestimated bacterial concentration compared to CCU50 and qPCR.•CCU50 detected M. ovipneumoniae viability, qPCR and flow cytometry did not.