Multilocus short sequence repeat sequencing approach for differentiating among Mycobacterium avium subsp. paratuberculosis strains

We describe a multilocus short sequence repeat (MLSSR) sequencing approach for the genotyping of Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) strains. Preliminary analysis identified 185 mono-, di-, and trinucleotide repeat sequences dispersed throughout the M. paratuberculosis genome, of which 78 were perfect repeats. Comparative nucleotide sequencing of the 78 loci of six M. paratuberculosis isolates from different host species and geographic locations identified a subset of 11 polymorphic short sequence repeats (SSRs), with an average of 3.2 alleles per locus. Comparative sequencing of these 11 loci was used to genotype a collection of 33 M. paratuberculosis isolates representing different multiplex PCR for IS900 loci (MPIL) or amplified fragment length polymorphism (AFLP) types. The analysis differentiated the 33 M. paratuberculosis isolates into 20 distinct MLSSR types, consistent with geographic and epidemiologic correlates and with an index of discrimination of 0.96. MLSSR analysis was also clearly able to distinguish between sheep and cattle isolates of M. paratuberculosis and easily and reproducibly differentiated strains representing the predominant MPIL genotype (genotype A18) and AFLP genotypes (genotypes Z1 and Z2) of M. paratuberculosis described previously. Taken together, the results of our studies suggest that MLSSR sequencing enables facile and reproducible high-resolution subtyping of M. paratuberculosis isolates for molecular epidemiologic and population genetic analyses.     

Molecular epidemiology of Mycobacterium avium subsp. paratuberculosis isolates recovered from wild animal species

Mycobacterial isolates were obtained by radiometric culture from 33 different species of captive or free-ranging animals (n = 106) and environmental sources (n = 3) from six geographic zones within the United States. The identities of all 109 isolates were confirmed by using mycobactin J dependence and characterization of five well-defined molecular markers, including two integration loci of IS900 (loci L1 and L9), one Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis)-specific sequence (locus 251), and one M. avium subsp. avium-specific marker (IS1245), as well as hsp65 and IS1311 restriction endonuclease analyses. Seventy-six acid-fast isolates were identified as M. paratuberculosis, 15 were identified as belonging to the M. avium-M. intracellulare complex (but not M. paratuberculosis), and the remaining 18 were identified as mycobacteria outside the M. avium-M. intracellulare complex. Fingerprinting by multiplex PCR for IS900 integration loci clustered 67 of the 76 M. paratuberculosis strains into a single clade (designated clade A18) and had a Simpson's diversity index (D) of 0.53. In contrast, sequence-based characterization of a recently identified M. paratuberculosis short sequence repeat (SSR) region enabled the differentiation of the M. paratuberculosis isolates in clade A18 into seven distinct alleles (D = 0.75). The analysis revealed eight subtypes among the 33 species of animals, suggesting the interspecies transmission of specific strains. Taken together, the results of our analyses demonstrate that SSR analysis enables the genetic characterization of M. paratuberculosis isolates from different host species and provide evidence for the host specificity of some M. paratuberculosis strains as well as sharing of strains between wild and domesticated animal species.     

Swine-to-Human Transmission of Influenza A(H3N2) Virus at Agricultural Fairs,Ohio, USA, 2012

Agricultural fairs provide an opportunity for bidirectional transmission of influenza A viruses. We sought to determine influenza A virus activity among swine at fairs in the United States. As part of an ongoing active influenza A virus surveillance project, nasal swab samples were collected from exhibition swine at 40 selected Ohio agricultural fairs during 2012. Influenza A(H3N2) virus was isolated from swine at 10 of the fairs. According to a concurrent public health investigation, 7 of the 10 fairs were epidemiologically linked to confirmed human infections with influenza A(H3N2) variant virus. Comparison of genome sequences of the subtype H3N2 isolates recovered from humans and swine from each fair revealed nucleotide identities of >99.7%, confirming zoonotic transmission between swine and humans. All influenza A(H3N2) viruses isolated in this study, regardless of host species or fair, were >99.5% identical, indicating that 1 virus strain was widely circulating among exhibition swine in Ohio during 2012.     

Cetylated fatty acids improve knee function in patients with osteoarthritis

OBJECTIVE: To determine the benefit of cetylated fatty acids (CFA) on knee range of motion and function in patients with osteoarthritis (OA). METHODS: Sixty-four patients with chronic knee OA were evaluated at baseline and at 30 and 68 days after consuming either placebo (vegetable oil; n = 31) or CFA (Celadrin; n = 33). Evaluations included physician assessment, knee range of motion with goniometry, and the Lequesne Algofunctional Index (LAI). RESULTS: After 68 days, patients treated with CFA exhibited significant (p < 0.001) increase in knee flexion (10.1 degrees) compared to patients given placebo (1.1 degrees). Neither group reported improvement in knee extension. Patient responses to the LAI indicated a significant (p < 0.001) shift towards functional improvement for the CFA group (-5.4 points) after 68 days compared to a modest improvement in the placebo group (-2.1 points). CONCLUSION: Compared to placebo, CFA provides an improvement in knee range of motion and overall function in patients with OA of the knee. CFA may be an alternative to the use of nonsteroidal antiinflammatory drugs for the treatment of OA.     

Human Escherichia coli O157:H7 genetic marker in isolates of bovine origin

The antiterminator Q gene of bacteriophage 933W (Q933) was identified upstream of the stx2 gene in 90% of human disease-origin Escherichia coli O157:H7 isolates and in 44.5% of bovine isolates. Shiga toxin production was higher in Q933-positive isolates than Q933-negative isolates. This genetic marker may provide a useful molecular tool for epidemiologic studies.     

Amplification of four genes of influenza A viruses using a degenerate primer set in a one step RT-PCR method

We designed a degenerate primer set that yielded full-length amplification of hemagglutinin (HA), neuraminidase (NA), matrix (M), and non-structural protein (NSP) genes of influenza A viruses in a single reaction mixture. These four genes were amplified from 15 HA (1–15) and 9 NA (1–9) subtypes of influenza A viruses of avian (n = 16) origin. In addition, 272 field isolates of avian origin were tested by this method. Full-length amplification of HA, NA, M, and NSP genes was obtained in 242 (88.9%), 254 (93.4%), 268 (98.5%), and 268 (98.5%) isolates, respectively. No gene was amplified in four isolates. Of these four isolates, two were subtyped as H4N6, one as H7N7, and one as H10N7. Amplification was successful for all 4 genes of H1N1, H2N3, and H3N2 isolates of swine influenza. Also, all four genes were amplified in one equine influenza (H3N8) isolate and seven isolates of human origin (H1N1 and H3N2). This appears to be the first study using degenerate primer set for full-length amplification of four genes of influenza A viruses in a single reaction. Further studies are needed to determine if this primer set can be used for subtyping of influenza virus isolates.     

Microbial shifts in the swine distal gut in response to the treatment with antimicrobial growth promoter,tylosin

Antimicrobials have been used extensively as growth promoters (AGPs) in agricultural animal production. However, the specific mechanism of action for AGPs has not yet been determined. The work presented here was to determine and characterize the microbiome of pigs receiving one AGP, tylosin, compared with untreated pigs. We hypothesized that AGPs exerted their growth promoting effect by altering gut microbial population composition. We determined the fecal microbiome of pigs receiving tylosin compared with untreated pigs using pyrosequencing of 16S rRNA gene libraries. The data showed microbial population shifts representing both microbial succession and changes in response to the use of tylosin. Quantitative and qualitative analyses of sequences showed that tylosin caused microbial population shifts in both abundant and less abundant species. Our results established a baseline upon which mechanisms of AGPs in regulation of health and growth of animals can be investigated. Furthermore, the data will aid in the identification of alternative strategies to improve animal health and consequently production.     

Efficient Differentiation of Mycobacterium avium Complex Species and Subspecies by Use of Five-Target Multiplex PCR

Infections caused by the Mycobacterium avium complex (MAC) are on the rise in both human and veterinary medicine. A means of effectively discriminating among closely related yet pathogenetically diverse members of the MAC would enable better diagnosis and treatment as well as further our understanding of the epidemiology of these pathogens. In this study, a five-target multiplex PCR designed to discriminate MAC organisms isolated from liquid culture media was developed. This MAC multiplex was designed to amplify a 16S rRNA gene target common to all Mycobacterium species, a chromosomal target called DT1 that is unique to M. avium subsp. avium serotypes 2 and 3, to M. avium subsp. silvaticum, and to M. intracellulare, and three insertion sequences, IS900, IS901, and IS1311. The pattern of amplification results allowed determination of whether isolates were mycobacteria, whether they were members of the MAC, and whether they belonged to one of three major MAC subspecies, M. avium subsp. paratuberculosis, M. avium subsp. avium, and M. avium subsp. hominissuis. Analytical sensitivity was 10 fg of M. avium subsp. paratuberculosis genomic DNA, 5 to 10 fg of M. avium subsp. avium genomic DNA, and 2 to 5 fg of DNA from other mycobacterial species. Identification accuracy of the MAC multiplex was evaluated by testing 53 bacterial reference strains consisting of 28 different mycobacterial species and 12 nonmycobacterial species. Identification accuracy in a clinical setting was evaluated for 223 clinical MAC isolates independently identified by other methods. Isolate identification agreement between the MAC multiplex and these comparison assays was 100%. The novel MAC multiplex is a rapid, reliable, and simple assay for discrimination of MAC species and subspecies in liquid culture media.Since the early 1980s, there has been an increase in disease caused by organisms broadly categorized as nontuberculous mycobacteria (NTM), a generic term for mycobacteria not in the Mycobacterium tuberculosis complex and other than M. leprae (32). Of these NTM, Mycobacterium avium complex (MAC) species are the most common cause of human and animal disease globally (6, 14, 16, 24). The clinical relevance of the MAC in humans has been amplified in recent decades with the increasing population of immunocompromised individuals resulting from longer life expectancy, immunosuppressive chemotherapy, and the AIDS pandemic (27). The MAC is divided into two main species: M. avium and M. intracellulare. M. avium is further subdivided (per Turenne et al.) into four subspecies: M. avium subsp. avium, M. avium subsp. hominissuis, M. avium subsp. paratuberculosis, and M. avium subsp. silvaticum (39).Members of the family Mycobacteriaceae, comprising the MAC, differ in virulence and ecology. Those designated M. avium subsp. hominissuis are genomically diverse, low-virulence, opportunistic pathogens for both animals and humans. The majority of human M. avium subsp. hominissuis infections occur in HIV-immunocompromised people, immunocompetent persons with underling pulmonary disease, and children with cystic fibrosis (2, 12, 17). Considered ubiquitous in the environment (the most likely source of infection for humans), M. avium subsp. hominissuis has been isolated from water, soil, and dust (9). Domestic water distribution systems have been reported as possible sources of M. avium subsp. hominissuis infections in hospitals, homes, and commercial buildings (26, 27). In animals, M. avium subsp. hominissuis is found as a cause of lymphadenitis of the head and mesenteric lymph nodes of swine recognized at slaughter.Mycobacterium avium subsp. avium has long been recognized as a primary pathogen causing avian tuberculosis in wild and domestic birds (37, 38). Members of this subspecies also sporadically cause disease in other animals (6, 15, 30).For veterinarians, the MAC member of greatest importance is M. avium subsp. paratuberculosis. This MAC member causes a chronic granulomatous enteritis called Johne''s disease or paratuberculosis, most often in ruminants (16, 22, 31). Mycobacterium avium subsp. paratuberculosis is capable of infecting and causing disease a wide array of animal species, including nonhuman primates, without need of immunosuppressive coinfections. The herd-level prevalence of M. avium subsp. paratuberculosis infections in dairy cattle exceeds 50% in most major dairy product-producing countries (29, 31). Two systematic reviews and meta-analyses report a consistent association of M. avium subsp. paratuberculosis with Crohn''s disease, and the zoonotic potential of M. avium subsp. paratuberculosis continues to be a controversial subject discussed in the literature (1, 11). Unlike for most other M. avium subspecies, isolation of M. avium subsp. paratuberculosis requires the addition of the siderophore mycobactin to culture media and prolonged culture incubation for successful isolation from a tissue, soil, or fecal samples (43). After this lengthy incubation period with special media, resultant acid-fast organisms then need to be accurately identified.Unlike the M. avium subspecies, whose type strains were obtained from nonhuman hosts, the type strain of M. intracellulare (ATCC 13950) was isolated from a human, specifically a child who died from disseminated disease. Recently, numerous isolates considered to be M. intracellulare were reclassified as M. chimaera sp. nov. as part of the MAC (35). Few of these isolates were found to be clinically relevant, suggesting that this MAC species has low pathogenicity, and this factor is crucial to therapeutic decision making. Mycobacterium intracellulare appears to have a distinct environmental niche, more prevalent in biofilms and at significantly higher CFU numbers than M. avium (10, 36). It accounts for more documented human infections than M. avium subsp. hominissuis in several countries, including South Korea and Japan (19, 20, 23).Contemporary methods for MAC identification, e.g., high-performance liquid chromatography (HPLC) of cell wall mycolic acids, and genetic probes based on rRNA targets, e.g., AccuProbe, cannot discriminate among M. avium subspecies (2, 9). Given the differences in pathogenicity among M. avium subspecies and the implications regarding the infection source, a practical and accurate method of simply identifying M. avium subspecies is needed (13, 25, 35). In this study, we describe the specificity, discrimination capacity, and sensitivity of a novel five-target PCR, called the MAC multiplex, using a wide array of reference and clinical MAC isolates and numerous nonmycobacterial organisms.