Our study demonstrates the repeated isolation of vaccine-derived Newcastle disease viruses from different species of wild birds across four continents from 1997 through 2014. The data indicate that at least 17 species from ten avian orders occupying different habitats excrete vaccine-derived Newcastle disease viruses. The most frequently reported isolates were detected among individuals in the order Columbiformes (n = 23), followed in frequency by the order Anseriformes (n = 13). Samples were isolated from both free-ranging (n = 47) and wild birds kept in captivity (n = 7). The number of recovered vaccine-derived viruses corresponded with the most widely utilized vaccines, LaSota (n = 28) and Hitchner B1 (n = 19). Other detected vaccine-derived viruses resembled the PHY-LMV2 and V4 vaccines, with five and two cases, respectively. These results and the ubiquitous and synanthropic nature of wild pigeons highlight their potential role as indicator species for the presence of Newcastle disease virus of low virulence in the environment. The reverse spillover of live agents from domestic animals to wildlife as a result of the expansion of livestock industries employing massive amounts of live virus vaccines represent an underappreciated and poorly studied effect of human activity on wildlife. The livestock-wildlife interface, historically underappreciated as a cause of disease emergence, is now recognized as an intersection from which pathogens can be transmitted from agricultural to free-ranging hosts, and vice versa [1] [2] [3] [4] . Some factors responsible for the presence of etiological agents at this interface include ecological changes such as spatial and temporal landuse alterations, pathogen adaptations to new hosts, and the introduction of non-native, permissive species [5] [6] [7] [8] . Of all the factors influencing disease emergence, likely the most substantial is the loss of ecological species barriers, permitting opportunistic pathogens access to wildlife [9] [10] [11] [12] [13] . Microbial bidirectional spillover between wildlife and domesticated species is a recognized but understudied event [10, 14, 15] . As an example, avian influenza is a biosecurity threat to the poultry industry due to its spillover from waterfowl reservoirs and subsequent viral spill back from domestic chickens (Gallus gallus) [16, 17] . What remains largely unstudied, however, is the spillover of live vaccines from domesticated species into wildlife. For the purpose of this paper we define "spillover or spillover of live vaccines" as the unintended transmission of live poultry vaccine viruses from domestic, gallinaceous poultry into non-target birds. Inadequate recognition of spillover events may be due to a lack of surveillance efforts, low mortality of infected animals, migration, or undetected resident wild bird morbidity [18] [19] [20] . The continuous expansion of the poultry industry, coupled with the mass employment of live virus vaccines, increases the probability of spillover of vaccines [21] [22] [23] . Examining the extent of spillover of live vaccines, including recently developed recombinant vaccines, from poultry into wild birds is crucial because the downstream epidemiological consequences of such spillovers are still unknown. Poultry producers routinely employ multiple live vaccines against economically significant pathogens, such as Marek's disease virus, Infectious bursal disease virus, Infectious bronchitis virus, Infectious laryngotracheitis virus, and Newcastle disease virus (NDV) [24] [25] [26] [27] . Circulating live vaccine viruses present additional risks such as reversion of virulence and recombination with wild-type strains. In addition, the immune response of wild birds induced by infection with vaccine strains may provide selective pressures resulting in viral antigenic drift or increased virulence [23, [28] [29] [30] [31] . Nonetheless, the use of live vaccines continues to dominate the poultry industry, as attested by their performance in the field. Their use is highly desirable as they are inexpensive, allow mass application, and stimulate both strong mucosal and cell-mediated immunity [32] [33] [34] . In the United States, the continuous demand for more effective vaccines is also likely driven by the industry's prior experience with large-scale poultry epidemics. For example, producers in California experienced Newcastle disease (ND) outbreaks in 1971 and again in 2002, each costing millions of dollars to eradicate and requiring the depopulation of millions of birds [35, 36] . Newcastle disease virus, also known as Avian paramyxovirus-1 (APMV-1), is a non-segmented, single-stranded RNA virus of the genus Avulavirus within the family Paramyxoviridae [37]. In particular, live vaccines against ND, a disease notifiable to the World Organization for Animal Health (OIE), are used both intensively and globally [38, 39] . The world poultry industry with tens of billions of commercial fowl is continuously expanding [40] . However, NDV also infect a broad range of free-ranging avifauna [41, 42] , notably Double-crested Cormorants (Phalacrocorax auritus) [43], dabbling ducks such as Mallards (Anas platyrynchos) [44], and even Adélie Penguins (Pygoscelis adeliae) from the South Shetland Islands [45]. Considering all of the aforementioned facts and the preliminary evidence of the presence of live vaccines in wild birds [46, 47] , we examined the hypothesis that NDV vaccines may spill into wild birds. First, we identified in GenBank databases existing cases of vaccine-derived NDV reported previously in wild birds [48] . Second, to expand upon those results, we also Spillover of Live NDV Vaccine Viruses PLOS ONE |
Output: Presence of Vaccine-Derived Newcastle Disease Viruses in Wild Birds a11111