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Low-pathogenic avian influenza viruses (LPAIVs) are genetically highly variable and have diversified into multiple evolutionary lineages that are primarily associated with wild-bird reservoirs. Antigenic variation has been described for mammalian influenza viruses and for highly pathogenic avian influenza viruses that circulate in poultry, but much less is known about antigenic variation of LPAIVs. In this study, we focused on H13 and H16 LPAIVs that circulate globally in gulls. We investigated the evolutionary history and intercontinental gene flow based on the hemagglutinin (HA) gene and used representative viruses from genetically distinct lineages to determine their antigenic properties by hemagglutination inhibition assays. For H13, at least three distinct genetic clades were evident, while for H16, at least two distinct genetic clades were evident. Twenty and ten events of intercontinental gene flow were identified for H13 and H16 viruses, respectively. At least two antigenic variants of H13 and at least one antigenic variant of H16 were identified. Amino acid positions in the HA protein that may be involved in the antigenic variation were inferred, and some of the positions were located near the receptor binding site of the HA protein, as they are in the HA protein of mammalian influenza A viruses. These findings suggest independent circulation of H13 and H16 subtypes in gull populations, as antigenic patterns do not overlap, and they contribute to the understanding of the genetic and antigenic variation of LPAIVs naturally circulating in wild birds.IMPORTANCE Wild birds play a major role in the epidemiology of low-pathogenic avian influenza viruses (LPAIVs), which are occasionally transmitted-directly or indirectly-from them to other species, including domestic animals, wild mammals, and humans, where they can cause subclinical to fatal disease. Despite a multitude of genetic studies, the antigenic variation of LPAIVs in wild birds is poorly understood. H

Low-pathogenic avian influenza viruses (LPAIVs) are genetically highly variable and have diversified into multiple evolutionary lineages that are primarily associated with wild-bird reservoirs. Antigenic variation has been described for mammalian influenza viruses and for highly pathogenic avian influenza viruses that circulate in poultry, but much less is known about antigenic variation of LPAIVs. In this study, we focused on H13 and H16 LPAIVs that circulate globally in gulls. We investigated the evolutionary history and intercontinental gene flow based on the hemagglutinin (HA) gene and used representative viruses from genetically distinct lineages to determine their antigenic properties by hemagglutination inhibition assays. For H13, at least three distinct genetic clades were evident, while for H16, at least two distinct genetic clades were evident. Twenty and ten events of intercontinental gene flow were identified for H13 and H16 viruses, respectively. At least two antigenic variants of H13 and at least one antigenic variant of H16 were identified. Amino acid positions in the HA protein that may be involved in the antigenic variation were inferred, and some of the positions were located near the receptor binding site of the HA protein, as they are in the HA protein of mammalian influenza A viruses. These findings suggest independent circulation of H13 and H16 subtypes in gull populations, as antigenic patterns do not overlap, and they contribute to the understanding of the genetic and antigenic variation of LPAIVs naturally circulating in wild birds. IMPORTANCE Wild birds play a major role in the epidemiology of low-pathogenic avian influenza viruses (LPAIVs), which are occasionally transmitted-directly or indirectly-from them to other species, including domestic animals, wild mammals, and humans, where they can cause subclinical to fatal disease. Despite a multitude of genetic studies, the antigenic variation of LPAIVs in wild birds is poorly understood. Her

The ability to maintain intra-cellular pH is crucial for bacteria and other microbes to survive in diverse environments, particularly those that undergo fluctuations in pH. Mechanisms of acid resistance remain poorly understood in mycobacteria. Although, studies investigating acid stress in M. tuberculosis are gaining traction, few center on Mycobacterium avium subsp. paratuberculosis (MAP), the etiological agent of chronic enteritis in ruminants. We identified a MAP acid stress response network involved in macrophage infection. The central node of this network was MAP0403, a predicted serine protease that shared an 86% amino acid identity with MarP in M. tuberculosis. Previous studies confirmed MarP as a serine protease integral to maintaining intra-bacterial pH and survival in acid in vitro and in vivo. We show that MAP0403 is upregulated in infected macrophages and MAC-T cells that coincided with phagosome acidification. Treatment of mammalian cells with bafilomcyin A1, a potent inhibitor of phagosomal vATPases, diminished MAP0403 transcription. MAP0403 expression was also noted in acidic medium. A surrogate host, M. smegmatis mc2 155, was designed to express MAP0403 and when exposed to either macrophages or in vitro acid stress had increased bacterial cell viability, which corresponds to maintenance of intra-bacterial pH in acidic (pH = 5) conditions, compared to the parent strain. These data suggest that MAP0403 may be the equivalent of MarP in MAP. Future studies confirming MAP0403 as a serine protease and exploring its structure and possible substrates are warranted.

Johne's disease or paratuberculosis, caused by Mycobacterium avium subsp. paratuberculosis (MAP), occurs in domestic and wild animals worldwide, causing a significant economic loss to livestock industries. After a prolonged incubation time, infected cattle shed MAP bacilli into feces and spread the disease to an uninfected animal population. It is largely unknown how (or whether) the interplay between the pathogen and the host immunity determines timing of shedding after the long incubation time. Such information would provide an understanding of pathogenesis in individual animals and the epidemiology of MAP infection in animal populations. In this review, we summarize current knowledge of bovine Johne's disease pathology, pathogenesis, immunology and genetics. We discuss knowledge gaps that direly need to be addressed to provide a science-based approach to diagnostics and (immuno)prophylaxis. These knowledge gaps are related to anatomical/clinical manifestation of MAP invasion, interaction of bacteria with phagocytes, granuloma formation, shedding, establishment and kinetics of adaptive immune responses in the pathogenesis of the disease. These topics are discussed at the molecular, cellular and tissue levels with special attention to the within host dynamics including the temporal and the spatial context relevant for the various host-pathogen interactions.

Johne’s disease or paratuberculosis, caused by Mycobacterium avium subsp. paratuberculosis (MAP), occurs in domesticand wild animals worldwide, causing a significant economic loss to livestock industries. After a prolonged incubationtime, infected cattle shed MAP bacilli into feces and spread the disease to an uninfected animal population. It is largelyunknown how (or whether) the interplay between the pathogen and the host immunity determines timing of sheddingafter the long incubation time. Such information would provide an understanding of pathogenesis in individual animalsand the epidemiology of MAP infection in animal populations. In this review, we summarize current knowledge ofbovine Johne’s disease pathology, pathogenesis, immunology and genetics. We discuss knowledge gaps that direlyneed to be addressed to provide a science-based approach to diagnostics and (immuno)prophylaxis. These knowledgegaps are related to anatomical/clinical manifestation of MAP invasion, interaction of bacteria with phagocytes, granulomaformation, shedding, establishment and kinetics of adaptive immune responses in the pathogenesis of the disease. Thesetopics are discussed at the molecular, cellular and tissue levels with special attention to the within host dynamicsincluding the temporal and the spatial context relevant for the various host-pathogen interactions.

Salmonella enterica is a leading cause of food borne illness. Recent studies have shown that S. enterica is a pathogen capable of causing alterations to the composition of the intestinal microbiome. A recent prospective study of French pork production farms found a statistically significant association between Lawsonia intracellularis and carriage of S. enterica. In the current study the composition of the gut microbiome was determined in pigs challenged with S. enterica serovar Typhimurium and or L. intracellularis and compared to non-challenged control pigs. Principal coordinate analysis demonstrated that there was a disruption in the composition of the gut microbiome in the colon and cecum of pigs challenged with either pathogen. The compositions of the microbiomes of challenged pigs were similar to each other but differed from the non-challenged controls. There also were statistically significant increases in Anaerobacter, Barnesiella, Pediococcus, Sporacetigenium, Turicibacter, Catenibacterium, Prevotella, Pseudobutyrivibrio, and Xylanibacter in the challenged pigs. To determine if these changes were specific to experimentally challenged pigs, we determined the compositions of the fecal microbiomes of naturally infected pigs that were carriers of S. enterica. Pigs that were frequent shedders of S. enterica were shown to have similar fecal microbiomes compared to non-shedders or pigs that shed S. enterica infrequently. In a comparison of the differentially abundant bacteria in the naturally infected pigs compared to experimentally challenged pigs, 9 genera were differentially abundant and each exhibited the same increase or decrease in abundance between the two groups. Thus, there were similar changes in the GI microbiome associated with carriage of S. enterica regardless of whether the pigs were experimentally challenged with S. enterica or acquired it naturally.

Vaccination remains a major tool for prevention and progression of Johne’s disease, a chronic enteritis of ruminants worldwide. Currently there is only one licensed vaccine within the United States and two vaccines licensed internationally against Johne’s disease. All licensed vaccines reduce fecal shedding of Mycobacterium avium subsp. paratuberculosis (MAP) and delay disease progression. However, there are no available vaccines that prevent disease onset. A joint effort by the Johne’s Disease Integrated Program (JDIP), a USDA-funded consortium, and USDA—APHIS/VS sought to identify transposon insertion mutant strains as vaccine candidates in part of a three phase study. The focus of the Phase I study was to evaluate MAP mutant attenuation in a well-defined in vitro bovine monocyte-derived macrophage (MDM) model. Attenuation was determined by colony forming unit (CFUs) counts and slope estimates. Based on CFU counts alone, the MDM model did not identify any mutant that significantly differed from the wild-type control, MAP K-10. Slope estimates using mixed models approach identified six mutants as being attenuated. These were enrolled in protection studies involving murine and baby goat vaccination-challenge models. MDM based approach identified trends in attenuation but this did not correlate with protection in a natural host model. These results suggest the need for alternative strategies for Johne’s disease vaccine candidate screening and evaluation.

Francisella tularensis, a Gram-negative bacterium and causative agent of tularemia, is categorized as a Class A select agent by the Centers for Disease Control and Prevention due to its ease of dissemination and ability to cause disease. Oropharyngeal and gastrointestinal tularemia may occur due to ingestion of contaminated food and water. Despite the concern to public health, little research is focused on F. tularensis detection in food and environmental matrices. Current diagnostics rely on host responses and amplification of F. tularensis genetic elements via Polymerase Chain Reaction; however, both tools are limited by development of an antibody response and limit of detection, respectively. During our investigation to develop an improved culture medium to aid F. tularensis diagnostics, we found enhanced F. tularensis growth using the spent culture filtrate. Addition of the spent culture filtrate allowed for increased detection of F. tularensis in mixed cultures of food and environmental matrices. Ultraperformance liquid chromatography (UPLC)/MS analysis identified several unique chemicals within the spent culture supernatant of which carnosine had a matching m/z ratio. Addition of 0.625 mg/mL of carnosine to conventional F. tularensis medium increased the growth of F. tularensis at low inoculums. In order to further enrich F. tularensis cells, we developed a DNA aptamer cocktail to physically separate F. tularensis from other bacteria present in food and environmental matrices. The combined enrichment steps resulted in a detection range of 1-106 CFU/mL (starting inoculums) in both soil and lettuce backgrounds. We propose that the two-step enrichment process may be utilized for easy field diagnostics and subtyping of suspected F. tularensis contamination as well as a tool to aid in basic research of F. tularensis ecology.

Defining genetic diversity in the wake of the release of several Mycobacterium avium subsp. paratuberculosis (MAP) genome sequences has become a major emphasis in the molecular biology and epidemiology of Johne’s disease research. These data can now be used to define the extent of strain diversity on the farm. However, to perform these important tasks, researchers must have a way to distinguish the many MAP isolates/strains that are present in the environment or host to enable tracking over time. Recent studies have described genetic diversity of the Mycobacterium avium complex (MAC), of which MAP is a member, through pulsed-field gel electrophoresis, single sequence repeats, variable-number tandem repeats, genome rearrangements, single nucleotide polymorphisms and genomewide comparisons to identify insertions and deletions. Combinations of these methods can now provide discrimination sufficient for dependable strain tracking. These molecular epidemiology techniques are being applied to understand transmission of Johne’s disease within dairy cattle herds as well as identify which strains predominate in wildlife.

Background: The genome of Mycobacterium avium subspecies paratuberculosis (MAP) is remarkably homogeneous among the genomes of bovine, human and wildlife isolates. However, previous work in our laboratories with the bovine K-10 strain has revealed substantial differences compared to sheep isolates. To systematically characterize all genomic differences that may be associated with the specific hosts, we sequenced the genomes of three U.S. sheep isolates and also obtained an optical map. Results: Our analysis of one of the isolates, MAP S397, revealed a genome 4.8 Mb in size with 4,700 open reading frames (ORFs). Comparative analysis of the MAP S397 isolate showed it acquired approximately 10 large sequence regions that are shared with the human M. avium subsp. hominissuis strain 104 and lost 2 large regions that are present in the bovine strain. In addition, optical mapping defined the presence of 7 large inversions between the bovine and ovine genomes (~ 2.36 Mb). Whole-genome sequencing of 2 additional sheep strains of MAP (JTC1074 and JTC7565) further confirmed genomic homogeneity of the sheep isolates despite the presence of polymorphisms on the nucleotide level. Conclusions: Comparative sequence analysis employed here provided a better understanding of the host association, evolution of members of the M. avium complex and could help in deciphering the phenotypic differences observed among sheep and cattle strains of MAP. A similar approach based on whole-genome sequencing combined with optical mapping could be employed to examine closely related pathogens. We propose an evolutionary scenario for M. avium complex strains based on these genome sequences.

The Standards for Reporting of Diagnostic Accuracy (STARD) statement (www.stard-statement.org) was developed to encourage complete and transparent reporting of key elements of test accuracy studies in human medicine. The statement was motivated by widespread evidence of bias in test accuracy studies and the finding that incomplete or absent reporting of items in the STARD checklist was associated with overly optimistic estimates of test performance characteristics. Although STARD principles apply broadly, specific guidelines do not exist to account for unique considerations in livestock studies such as herd tests, potential use of experimental challenge studies, a more diverse group of testing purposes and sampling designs, and the widespread lack of an ante-mortem reference standard with high sensitivity and specificity. The objective of the present study was to develop a modified version of STARD relevant to paratuberculosis (Johne's disease) in ruminants. Examples and elaborations for each of the 25 items were developed by a panel of experts using a consensus-based approach to explain the items and underlying concepts. The new guidelines, termed STRADAS-paraTB (Standards for Reporting of Animal Diagnostic Accuracy Studies for paratuberculosis), should facilitate improved quality of reporting of the design, conduct and results of paratuberculosis test accuracy studies which were identified as “poor” in a review published in 2008 in Veterinary Microbiology

The Standards for Reporting of Diagnostic Accuracy (STARD) statement (www.stard-statement.org) was developed to encourage complete and transparent reporting of key elements of test accuracy studies in human medicine. The statement was motivated by widespread evidence of bias in test accuracy studies and the finding that incomplete or absent reporting of items in the STARD checklist was associated with overly optimistic estimates of test performance characteristics. Although STARD principles apply broadly, specific guidelines do not exist to account for unique considerations in livestock studies such as herd tests, potential use of experimental challenge studies, a more diverse group of testing purposes and sampling designs, and the widespread lack of an ante-mortem reference standard with high sensitivity and specificity. The objective of the present study was to develop a modified version of STARD relevant to paratuberculosis (Johne's disease) in ruminants. Examples and elaborations for each of the 25 items were developed by a panel of experts using a consensus-based approach to explain the items and underlying concepts. The new guidelines, termed STRADAS-paraTB (Standards for Reporting of Animal Diagnostic Accuracy Studies for paratuberculosis), should facilitate improved quality of reporting of the design, conduct and results of paratuberculosis test accuracy studies which were identified as “poor” in a review published in 2008 in Veterinary Microbiology

The Standards for Reporting of Diagnostic Accuracy (STARD) statement (www.stard-statement.org) was developed to encourage complete and transparent reporting of key elements of test accuracy studies in human medicine. The statement was motivated by widespread evidence of bias in test accuracy studies and the finding that incomplete or absent reporting of items in the STARD checklist was associated with overly optimistic estimates of test performance characteristics. Although STARD principles apply broadly, specific guidelines do not exist to account for unique considerations in livestock studies such as herd tests, potential use of experimental challenge studies, a more diverse group of testing purposes and sampling designs, and the widespread lack of an ante-mortem reference standard with high sensitivity and specificity. The objective of the present study was to develop a modified version of STARD relevant to paratuberculosis (Johne's disease) in ruminants. Examples and elaborations for each of the 25 items were developed by a panel of experts using a consensus-based approach to explain the items and underlying concepts. The new guidelines, termed STRADAS-paraTB (Standards for Reporting of Animal Diagnostic Accuracy Studies for paratuberculosis), should facilitate improved quality of reporting of the design, conduct and results of paratuberculosis test accuracy studies which were identified as “poor” in a review published in 2008 in Veterinary Microbiology

Due to a close genetic relatedness, there is no known antibody that detects Mycobacterium avium subspecies paratuberculosis (MAP), which causes Johne’s disease in cattle and sheep, and does not cross-react with other M. avium subspecies. In the present study, a monoclonal antibody (MAb; 17A12) was identified from mice immunized with a cell membrane fraction of MAP strain K-10. This antibody is 100% specific as it detected a 25-kDa protein in all 29 MAP whole cell lysates, but did not bind to any of the 29 non- paratuberculosis strains tested in immunoblot assays. However, the antibody revealed variable reactivity levels in MAP strains as it detected higher levels in bovine isolates but comparably lower levels in ovine isolates of MAP. In order to identify the target binding protein for 17A12, a lambda phage expression library of MAP genomic fragments was screened with the MAb. Four reactive clones were identified, sequenced and all shown to be overlapping. Further analysis revealed all four clones expressed an unknown protein encoded by a sequence that is not annotated in the K-10 genome and overlapped with MAP3422c on the opposing DNA strand. The epitope of 17A12 was precisely defined to seven amino acids and was used to query the K-10 genome. Similarity searches revealed another protein, encoded by MAP1025, possessed a similar epitope (one-amino acid mis- match) that also reacted strongly to the antibody. A single nucleotide polymorphism (SNP) in MAP1025 was then identified by comparative sequence analysis, which results in a Pro28His change at residue 28, the first amino acid within the 17A12 epitope. This SNP is present in all MAP strains but absent in all non-MAP strains and accounts for the speci- ficity of the 17A12 antibody. This new antibody is the first ever isolated that binds only to the paratuberculosis subspecies of M. avium and opens new possibilities for the specific detection of this significant ruminant pathogen.