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Two independent strains of a Leptotrichia species (ES3154-GLU T and ES2714_GLU) were isolated from the oral cavity of northern elephant seals ( Mirounga angustirostris ) that were admitted to The Marine Mammal Centre facilities in California, USA. The strains were isolated from oral swabs by cultivation in PPLO broth supplemented with serum, penicillin and colistin in anaerobic conditions. The strains were Gram-negative, pleomorphic, indole-, oxidase- and catalase-negative, non-spore-forming, non-motile rods/coccobacilli in short chains. The 16S rRNA gene sequence of these strains shared 94.42 % nucleotide similarity with Oceanivirga salmonicida AVG 2115 T but demonstrated ≤86.00-92.50 % nucleotide similarity to the 16S rRNA genes of other species of the family Leptotrichiaceae . The genome was sequenced for strain ES3154-GLU T . Average nucleotide identity values between strain ES3154-GLU T and 15 type strain genomes from the family Leptotrichiaceae ranged from 66.74 % vs. Sebaldella termitidis to 73.35 % vs. O. salmonicida . The whole genome phylogeny revealed that the novel species was most closely related to O. salmonicida AVG 2115 T . This relationship was also confirmed by nucleotide similarity and multilocus phylogenetic analyses employing various housekeeping genes (partial 23S rRNA, rpoB , rpoC , rpoD , polC , adh , gyrA and gyrB genes). Chemotaxonomic and phenotypical features of strain ES3154-GLU T were in congruence with closely related members of the family Leptotrichiaceae , represented by similar enzyme profiles and fatty acid patterns. MALDI-TOF MS analysis was capable to clearly discriminate strain ES3154-GLU T from all currently described taxa of the family Leptotrichiaceae . Based on these data, we propose a novel species of the genus Oceanivirga , for which the name Oceanivirga miroungae sp. nov. is proposed with the type strain ES3154-GLU T (=DSM 109740 T =CCUG 73653 T =ATCC TSD-189 T =NCTC 14411 T ) and one representative strain ES2714_GLU. The G+C content is 26.82 %, genome size is 1 356 983 bp.

In 1983, Mycoplasma sp. strain 1220 was isolated in Hungary from the phallus lymph of a gander with phallus inflammation. Between 1983 and 2017, Mycoplasma sp. 1220 was also identified and isolated from the respiratory tract, liver, ovary, testis, peritoneum and cloaca of diseased geese in several countries. Seventeen studied strains produced acid from glucose and fructose but did not hydrolyse arginine or urea, and all grew under aerobic, microaerophilic and anaerobic conditions at 35 to 37 ˚C in either SP4 or pleuropneumonia-like organism medium supplemented with glucose and serum. Colonies on agar showed a typical fried-egg appearance and transmission electron microscopy revealed a typical mycoplasma cellular morphology. Molecular characterization included analysis of the following genetic loci: 16S rRNA, 23S rRNA, 16S-23S rRNA ITS, rpoB , rpoC , rpoD , uvrA , parC , topA , dnaE , fusA and pyk . The genome was sequenced for type strain 1220 T . The 16S rRNA gene sequences of studied strains of Mycoplasma sp. 1220 shared 99.02-99.19 % nucleotide similarity with M. anatis strains but demonstrated ≤95.00-96.70 % nucleotide similarity to the 16S rRNA genes of other species of the genus Mycoplasma . Phylogenetic, average nucleotide and amino acid identity analyses revealed that the novel species was most closely related to Mycoplasma anatis . Based on the genetic data, we propose a novel species of the genus Mycoplasma , for which the name Mycoplasma anserisalpingitidis sp. nov. is proposed with the type strain 1220 T (=ATCC BAA-2147 T =NCTC 13513 T =DSM 23982 T ). The G+C content is 26.70 mol%, genome size is 959110 bp.

Most emerging pathogens can infect multiple species, underlining the importance of understanding the ecological and evolutionary factors that allow some hosts to harbour greater infection prevalence and share pathogens with other species. However, our understanding of pathogen jumps is based primarily around viruses, despite bacteria accounting for the greatest proportion of zoonoses. Because bacterial pathogens in bats (order Chiroptera) can have conservation and human health consequences, studies that examine the ecological and evolutionary drivers of bacterial prevalence and barriers to pathogen sharing are crucially needed. Here were studied haemotropic Mycoplasma spp. (i.e., haemoplasmas) across a species-rich bat community in Belize over two years. Across 469 bats spanning 33 species, half of individuals and two-thirds of species were haemoplasma positive. Infection prevalence was higher for males and for species with larger body mass and colony sizes. Haemoplasmas displayed high genetic diversity (21 novel genotypes) and strong host specificity. Evolutionary patterns supported codivergence of bats and bacterial genotypes alongside phylogenetically constrained host shifts. Bat species centrality to the network of shared haemoplasma genotypes was phylogenetically clustered and unrelated to prevalence, further suggesting rare-but detectable-bacterial sharing between species. Our study highlights the importance of using fine phylogenetic scales when assessing host specificity and suggests phylogenetic similarity may play a key role in host shifts not only for viruses but also for bacteria. Such work more broadly contributes to increasing efforts to understand cross-species transmission and the epidemiological consequences of bacterial pathogens., (© 2020 The Authors. Molecular Ecology published by John Wiley & Sons Ltd.)

We describe two novel species of Acholeplasma sp. strain N93 and Mycoplasma sp. strain LR5794 which were isolated from the nasopharynx of a horse from the United Kingdom and from the oral cavity of a North American raccoon from Canada, respectively. These strains were phenotypically and genetically characterized and compared to other established Mycoplasma and Acholeplasma species. Both strains are facultative anaerobes, resistant to penicillin, and produce acid from glucose but do not hydrolyze arginine and urea. Both strains grew well in microaerophilic and anaerobic atmospheric conditions at 35-37 °C using PPLO (pleuropneumonia-like organisms) medium. Acholeplasma sp. N93 does not require serum for growth. Colonies of both strains showed a typical fried-egg appearance and transmission electron microscopy of bacterial cells revealed a typical mycoplasma cellular morphology. Molecular characterization included assessment of several genetic loci. The genetic analysis indicated that Acholeplasma sp. N93 and Mycoplasma sp. LR5794 were most closely related to A. hippikon and A. equifetale, and M. molare and M. lagogenitalium, respectively. However, both novel strains were genetically unique in comparison to other well-known Mycoplasma and Acholeplasma species. Based on the isolation source history, phenotypic, genotypic, and phylogenetic characteristics of these novel strains, we propose the name Acholeplasma equirhinis sp. nov. for Acholeplasma sp. isolated from the nasopharynx of a horse [the type strain is N93 T (= DSM 106692 T  = ATCC TSD-139 T  = NCTC 14351 T )], and the name Mycoplasma procyoni sp. nov. for the Mycoplasma sp. isolated from the oral cavity of a North American raccoon [the type strain is LR5794 T (= DSM 106703 T  = ATCC TSD-141 T  = NCTC 14309 T )].

Novel ureaplasma strains have been isolated from the genital tract of both sexes of northern elephant seals ( Mirounga angustirostris ; six strains) and California sea lions ( Zalophus californianus ; five strains) stranded along the Central California coast, USA. These strains were phenotypically and genetically characterized and compared to other seven known Ureaplasma species. All novel ureaplasma strains hydrolysed urea, but did not metabolize arginine, and all were isolated and propagated using PPLO medium supplemented with urea under aerobic, microaerophilic, and anaerobic atmospheric conditions at +35-37 °C. Transmission electron microscopy revealed typical mollicute cellular morphology. Molecular characterization included assessment of the following genetic loci: 16S rRNA, the 16S-23S ITS, 23S rRNA, rpoB , ftsH , tufB , rpoC , fusA and ureC . Complete 16S rRNA gene sequence analysis of these novel Ureaplasma species indicated that they were most closely related to each other with nucleotide identity 99.87 % and ≤93.08 % related to other known Ureaplasma species. The results of nucleotide analysis of the sequenced housekeeping genes revealed 71.68-93.02 % similarity to corresponding genes of other known Ureaplasma species. The multi-locus genetic characterization and the phylogenetic analysis of the 16S rRNA and rpoB genes of these Ureaplasma species clearly demonstrated their novelty and, reflecting their host specificites, the name Ureaplasma miroungigenitalium sp. nov. is proposed for the Ureaplasma species isolated from northern elephant seals, the type strain is ES2783-GEN T (=DSM 24842 T =ATCC BAA-2460 T ), and the name Ureaplasma zalophigenitalium sp. nov. is proposed for the Ureaplasma species isolated from California sea lions, the type strain is CSL7644-GEN T (=DSM 24843 T =ATCC BAA-2262 T ).

Five Mycoplasma strains have been isolated from the oropharynx of southern sea otters (Enhydra lutris nereis) from the Central California Coast, USA. These strains were phenotypically and genetically characterized and compared to other established Mycoplasma species. All five strains hydrolysed arginine but not urea, but did not produce acid from glucose, and all were isolated and propagated under anaerobic and aerobic atmospheric conditions at +35-37 ˚C using either SP4 or PPLO medium supplemented with arginine. Colonies on solid medium showed a typical fried-egg appearance and transmission electron microscopy revealed a typical mycoplasma cellular morphology. Molecular characterization included assessment of the following genetic loci: 16S rRNA, 16S-23S rRNA ITS, rpoB, rpoC, polC, topIIA, tufB, arcA and smc. Complete 16S rRNA gene sequence analysis indicated that these strains were most closely related to M.ycoplasma phocicerebrale, and to M.ycoplasma arginini, M.ycoplasma gateae and M.ycoplasma canadense with nucleotide similarities of 99 and 98 %, respectively. Nucleotide analysis of other genetic loci revealed 73-91 % nucleotide similarity to the corresponding genes of the above closely related species. All five strains clustered into a distinct group on the 16S rRNA and rpoB phylogenetic trees. Serological testing via growth inhibition and metabolic inhibition tests employing antiserum to type strains of M. phocicerebrale, M. arginini, M. gateae and M. canadense failed to recognize these novel strains. Our results suggest that the strains isolated from southern sea otters represent a novel species of the genus Mycoplasma, for which the name Mycoplasma enhydrae sp. nov. is proposed; the type strain is 6243-11 T (=DSM 106704 T =ATCC TSD-140 T ).

Three independent strains of Neisseria sp. were isolated from the oral cavity of California sea lions (Zalophus californianus) that were admitted to The Marine Mammal Center facilities in California, USA. The strains were isolated from oral swabs by cultivation on Trypticase Soy agar with 5% sheep blood under aerobic conditions. The 16S rRNA gene sequence of these three strains shared 99% similarity, but demonstrated only 97-98% nucleotide similarity to the phylogenetically closest relatives such as N. canis, N. zoodegmatis, N. animaloris, and N. dumasiana. These three strains also shared 99% sequence similarity of their rplF, rpoB, and gyrB gene sequences. Based on the biochemical tests alone (i.e., without genetic analysis of housekeeping genes), it is difficult to discriminate this novel species from N. canis; however, it can be easily discriminated from all phylogenetically closely related species using the sequencing analysis of its housekeeping genes (e.g., rplF, rpoB, or gyrB genes). Thus, genetic testing is indispensable for accurate identification of this species in a routine laboratory practice. The species is an obligate aerobe and able to grow in Mueller-Hinton broth supplemented with 6% NaCl, but the phylogenetically closely related species (N. canis, N. zoodegmatis, N. animaloris, and N. dumasiana) were not. Based on these phenotypic and genotypic characteristics and phylogenetic data, we conclude that these new strains represent a novel species of the genus Neisseria, for which the name Neisseria zalophi sp. nov. is proposed. The type strain is CSL 7565 T (= ATCC BAA2455 T  = DSM 102031 T ).

Human activities create novel food resources that can alter wildlife-pathogen interactions. If resources amplify or dampen, pathogen transmission probably depends on both host ecology and pathogen biology, but studies that measure responses to provisioning across both scales are rare. We tested these relationships with a 4-year study of 369 common vampire bats across 10 sites in Peru and Belize that differ in the abundance of livestock, an important anthropogenic food source. We quantified innate and adaptive immunity from bats and assessed infection with two common bacteria. We predicted that abundant livestock could reduce starvation and foraging effort, allowing for greater investments in immunity. Bats from high-livestock sites had higher microbicidal activity and proportions of neutrophils but lower immunoglobulin G and proportions of lymphocytes, suggesting more investment in innate relative to adaptive immunity and either greater chronic stress or pathogen exposure. This relationship was most pronounced in reproductive bats, which were also more common in high-livestock sites, suggesting feedbacks between demographic correlates of provisioning and immunity. Infection with both Bartonella and haemoplasmas were correlated with similar immune profiles, and both pathogens tended to be less prevalent in high-livestock sites, although effects were weaker for haemoplasmas. These differing responses to provisioning might therefore reflect distinct transmission processes. Predicting how provisioning alters host-pathogen interactions requires considering how both within-host processes and transmission modes respond to resource shifts.This article is part of the theme issue 'Anthropogenic resource subsidies and host-parasite dynamics in wildlife'., (© 2018 The Authors.)

Bats (Order: Chiroptera) have been widely studied as reservoir hosts for viruses of concern for human and animal health. However, whether bats are equally competent hosts of non-viral pathogens such as bacteria remains an important open question. Here, we surveyed blood and saliva samples of vampire bats from Peru and Belize for hemotropic Mycoplasma spp. (hemoplasmas), bacteria that can cause inapparent infection or anemia in hosts. 16S rRNA gene amplification of blood showed 67% (150/223) of common vampire bats (Desmodus rotundus) were infected by hemoplasmas. Sequencing of the 16S rRNA gene amplicons revealed three novel genotypes that were phylogenetically related but not identical to hemoplasmas described from other (non-vampire) bat species, rodents, humans, and non-human primates. Hemoplasma prevalence in vampire bats was highest in non-reproductive and young individuals, did not differ by country, and was relatively stable over time (i.e., endemic). Metagenomics from pooled D. rotundus saliva from Peru detected non-hemotropic Mycoplasma species and hemoplasma genotypes phylogenetically similar to those identified in blood, providing indirect evidence for potential direct transmission of hemoplasmas through biting or social contacts. This study demonstrates vampire bats host several novel hemoplasmas and sheds light on risk factors for infection and basic transmission routes. Given the high frequency of direct contacts that arise when vampire bats feed on humans, domestic animals, and wildlife, the potential of these bacteria to be transmitted between species should be investigated in future work.

Raccoons ( Procyon lotor ) are successful urban adapters and hosts to a number of zoonotic and nonzoonotic pathogens, yet little is known about their hemoplasma infections and how prevalence varies across habitat types. This study identifies hemotropic Mycoplasma species infection in raccoons from urban and undisturbed habitats and compares hemoplasma infection in sympatric urban cats ( Felis catus ) from the same geographic region. We collected blood from raccoons ( n = 95) on an urban coastal island ( n = 37) and an undisturbed coastal island ( n = 58) and from sympatric urban cats ( n = 39) in Georgia, USA. Based on 16S rRNA gene amplification, 62.1% (59/95) of raccoons and 17.9% (7/39) of feral cats were positive for hemoplasma. There was a greater percentage of hemoplasma-infected raccoons on the undisturbed island (79.3% [46/58]) than on the urban island (35.1% [13/37]; χ 2 = 16.9, df = 1, P = 0.00004). Sequencing of the full-length 16S rRNA gene amplicons revealed six hemoplasma genotypes in raccoons, including five novel genotypes that were distinct from three known hemoplasma species identified in the sympatric cats. In addition, the hemoplasma genotypes detected in raccoons were not identified in sympatric cats or vice versa. Although all six hemoplasma genotypes were found in raccoons from urban and undisturbed islands, coinfection patterns differed between sites and among individuals, with the proportion of coinfected raccoons being greater in the undisturbed site. This study shows that raccoons are hosts for several novel hemoplasmas and that habitat type influences infection patterns. IMPORTANCE This study provides information about novel hemoplasmas identified in raccoons ( Procyon lotor ), which can be used for assessments of the prevalence of these hemoplasmas in raccoon populations and for future studies on the potential pathogenic impacts of these hemoplasmas on raccoon health. Raccoons from the undisturbed habitat had a higher prevalence of hemoplasma infection than urban raccoons. There does not appear to be cross-species transmission of hemotropic mycoplasmas between urban raccoons and feral cats. Raccoons appear to be hosts for several novel hemoplasmas, and habitat type influences infection patterns., (Copyright © 2017 American Society for Microbiology.)

Three strains of lactic acid bacteria (LAB) were isolated from the faeces of apparently healthy wild Canada geese (Branta canadensis) in 2010 by cultivating faecal LAB on Rogosa SL agar under aerobic conditions. These three isolates were found to share 99.9 % gene sequence similarity of their 16S rRNA, their 16S-23S intergenic transcribed spacer region (ITS), partial 23S rRNA, rpoB, rpoC, rpoA and pheS gene sequences. However, the three strains exhibited lower levels of sequence similarity of these genetic targets to all known LAB, and the phylogenetically closest species to the geese strains were Lactobacillus casei, Lactobacillus paracasei, Lactobacillus rhamnosus and Lactobacillus saniviri. In comparison to L. casei ATCC 393(T), L. paracasei ATCC 25302(T), L. rhamnosus ATCC 7469(T) and L. saniviri DSM 24301(T), the novel isolates reacted uniquely in tests for cellobiose, galactose, mannitol, citric acid, aesculin and dextrin, and gave negative results in tests for l-proline arylamidase and l-pyrrolydonyl-arylamidase, and in the Voges-Proskauer test. Biochemical tests for cellobiose, aesculin, galactose, gentiobiose, mannitol, melezitose, ribose, salicin, sucrose, trehalose, raffinose, turanose, amygdalin and arbutin could be used for differentiation between L. saniviri and the novel strains. On the basis of phenotypic and genotypic characteristics, and phylogenetic data, the three isolates represent a novel species of the genus Lactobacillus, for which the name Lactobacillus brantae sp. nov. is proposed. The type strain is SL1108(T) (= ATCC BAA-2142(T) = LMG 26001(T) = DSM 23927(T)) and two additional strains are SL1170 and SL60106.

Eosinophilic fasciitis (EF) with generalized sclerodermiform skin lesions developed over a 19-month period in a previously healthy 23-year-old man. Although we confirmed EF by skin histology and laboratory tests, the recurrent fevers and the clinical observation of sclerotic prepuce with urethritis indicated further bacteriological analysis by conventional microbiological and DNA-based tests. Urethra cultures were positive for an arginine-hydrolyzing mycoplasma and Ureaplasma urealyticum. The patient also had serum IgM antibodies to Mycoplasma pneumoniae using enzyme-linked immunosorbent assay (ELISA)-based qualitative detection. Mycoplasma arginini was isolated from two independent venous blood serum samples and was identified by conventional microbiological tests and sequencing of the 16S rRNA and rpoB genes (GenBank sequence accession numbers HM179555 and HM179556, respectively). M. arginini genomic DNA also was detected by species-specific PCR in the skin lesion biopsy sample. Treatment with corticosteroids and long-term courses of selected antibiotics led to remission of skin symptoms and normalization of laboratory values. This report provides the first evidence of EF associated with mycoplasma infection and the second report of human infection with M. arginini and therefore suggests that this mycoplasma infection might have contributed to the pathogenesis of the disease.

Conventional classification of the species in the family Mycoplasmataceae is mainly based on phenotypic criteria, which are complicated, can be difficult to measure, and have the potential to be hampered by phenotypic deviations among the isolates. The number of biochemical reactions suitable for phenotypic characterization of the Mycoplasmataceae is also very limited and therefore the strategy for the final identification of the Mycoplasmataceae species is based on comparative serological results. However, serological testing of the Mycoplasmataceae species requires a performance panel of hyperimmune sera which contains anti-serum to each known species of the family, a high level of technical expertise, and can only be properly performed by mycoplasma-reference laboratories. In addition, the existence of uncultivated and fastidious Mycoplasmataceae species/isolates in clinical materials significantly complicates, or even makes impossible, the application of conventional bacteriological tests. The analysis of available genetic markers is an additional approach for the primary identification and phylogenetic classification of cultivable species and uncultivable or fastidious organisms in standard microbiological laboratories. The partial nucleotide sequences of the RNA polymerase β-subunit gene (rpoB) and the 16S-23S rRNA intergenic transcribed spacer (ITS) were determined for all known type strains and the available non-type strains of the Mycoplasmataceae species. In addition to the available 16S rRNA gene data, the ITS and rpoB sequences were used to infer phylogenetic relationships among these species and to enable identification of the Mycoplasmataceae isolates to the species level. The comparison of the ITS and rpoB phylogenetic trees with the 16S rRNA reference phylogenetic tree revealed a similar clustering patterns for the Mycoplasmataceae species, with minor discrepancies for a few species that demonstrated higher divergence of their ITS and rpoB in comparison to their neighbor species. Overall, our results demonstrated that the ITS and rpoB gene could be useful complementary phylogenetic markers to infer phylogenetic relationships among the Mycoplasmataceae species and provide useful background information for the choice of appropriate metabolic and serological tests for the final classification of isolates. In summary, three-target sequence analysis, which includes the ITS, rpoB, and 16S rRNA genes, was demonstrated to be a reliable and useful taxonomic tool for the species differentiation within the family Mycoplasmataceae based on their phylogenetic relatedness and pairwise sequence similarities. We believe that this approach might also become a valuable tool for routine analysis and primary identification of new isolates in medical and veterinary microbiological laboratories., (Published by Elsevier Inc.)

The hemoplasmas are the trivial name for a group of erythrocyte-parasitizing, non-cultivable in vitro bacteria of the genus Mycoplasma that have been described in several mammalian hosts worldwide. This study is the first report of hemoplasmas in marine mammals. EDTA anticoagulated whole blood samples from 137 California sea lions (Zalophus californianus) and 20 northern elephant seals (Mirounga angustirostris) admitted to the Marine Mammal Center (Sausalito, CA; www.marinemammalcenter.org) or live captured in Oregon were collected during 2008. Hemoplasma-specific genomic DNA was detected in blood samples from 12.4% California sea lions tested using PCR. Hemoplasma PCR positive blood specimens also were tested in reverse transcription polymerase chain reaction (RT-PCR) using the hemoplasma-specific primers for the 16S and 23S rRNA genes. The RT-PCR showed the presence the hemoplasmal rRNA, strongly suggesting the presence of potentially viable hemoplasmas in the bloodstream of the animals. BLAST search and phylogenetic analysis of the 16S rRNA sequences of the hemoplasma from California sea lions revealed that the organism is a novel hemoplasma species with only 92.1% of its nucleotide similarity to the 16S rRNA gene of the previously described hemoplasma species of alpacas, Candidatus Mycoplasma haemolamae. Thus, due to low level of genetic similarity of the hemoplasma to other described hemoplasmas and the mammalian host in which the hemoplasma was detected we propose that this novel hemoplasma species has been given the provisional name Candidatus Mycoplasma haemozalophi sp. nov., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Mycoplasmas, particularly species of the genera Mycoplasma and Acholeplasma, are known to be occasional microbial contaminants of cell cultures that produce biologics. This presents a serious concern regarding the risk of mycoplasma contamination for research laboratories and commercial facilities developing and manufacturing cell-derived biological and biopharmaceutical products for therapeutic use. Potential undetected contamination of these products or process intermediates with mycoplasmas represents a potential safety risk for patients and a business risk for producers of biopharmaceuticals. To minimize these risks, monitoring for adventitious agents, such as viruses and mycoplasmas, is performed during the manufacture of biologics produced in cell culture substrates. The "gold standard" microbiological assay, currently recommended by the USP, EP, JP and the US FDA, for the mycoplasma testing of biologics, involves the culture of viable mycoplasmas in broth, agar plates and indicator cells. Although the procedure enables highly efficient mycoplasma detection in cell substrates and cell-derived products, the overall testing strategy is time consuming (a minimum of 28 days) and requires skilled interpretation of the results. The long time period required for these conventional assays does not permit their use for products with short shelf-lives or for timely 'go/no-go' decisions during routine in-process testing. PCR methodology has existed for decades, however PCR based and other alternative methods for mycoplasma detection have only recently been considered for application to biologics manufacture. The application of alternative nucleic acid-based, enzyme-based and/or recombinant cell-culture methods, particularly in combination with efficient sample preparation procedures, could provide advantages over conventional microbiological methods in terms of analytical throughput, simplicity, and turnaround time. However, a challenge to the application of alternative methods for detection of mycoplasmas remains whether these alternative methods can provide a limit of detection comparable or superior to those of the culture methods. An additional challenge is that nucleic acid amplification technique (NAT) methods do not allow for accurate discrimination between viable and non-viable mycoplasma contaminants, which might lead to false-positive results (e.g. from inactivated raw materials, etc.). Our review provides an overview of these alternative methods and discusses the pros and cons of their application for the testing of mycoplasmas in biologics and cell substrates., (Published by Elsevier Ltd.)

Microarrays are spatially ordered arrays with ligands chemically immobilized in discrete spots on a solid matrix, usually a microscope slide. Microarrays are a high-throughput large-scale screening system enabling simultaneous identification of a large number of labeled target molecules (up to several hundred thousand) that bind specifically to the immobilized ligands of the array. DNA microarrays represent a promising tool for clinical, environmental, and industrial microbiology since the technology allows relatively rapid identification of large number of genetic determinants simultaneously, providing detailed genomic level information regarding the pathogen species, including identification of their virulence-associated factors and the presence of antibiotic resistance genes. In this chapter, we describe key aspects and methodologies important for the development and use of DNA microarrays for microbial diagnostics.

The increasing emergence of drug-resistant Mycobacterium tuberculosis poses significant threat to the treatment of tuberculosis (TB). Conventional drug susceptibility testing is time-consuming and takes several weeks because of the slow growth rate of M. tuberculosis and the requirement for the drugs to show antimycobacterial activity. Resistance to TB drugs in M. tuberculosis is caused by mutations in the corresponding drug resistance genes (e.g., katG, inhA, rpoB, pncA, embB, rrs, gyrA, gyrB), and detection of these mutations can be a molecular indicator of drug resistance. In this chapter, we describe the utility of a microarray-based approach exploiting short overlapping oligonucleotides (sliding-frame array) to rapidly detect drug resistance-associated mutations (substitutions, deletions, and insertions) in the pncA gene responsible for resistance ofM. tuberculosis to pyrazinamide (PZA) as an example for this approach. Hybridization of pncA-derived RNA or DNA with the microarray enables easy and simple screening of nucleotide changes in the pncA gene. Sliding-frame microarrays can be used to identify other drug-resistant TB strains that have mutations in relevant drug resistance genes.

In this study, we describe our results on the evaluation of the ability of different permissive mammalian cell lines to support the biological enrichment of mycoplasma species known to be bacterial contaminants of cell substrates. The study showed that this approach is able to significantly improve the efficiency of mycoplasma detection based on nucleic acid testing or biochemical technologies (e.g., MycoAlert mycoplasma detection). Of 10 different cell lines (Vero, MDBK, HEK-293, Hep-G2, CV-1, EBTr, WI-38, R9ab, MDCK, and High Five) used in the study, only MDCK cell culture was found to support the efficient growth of all the tested mycoplasmas (Mycoplasma arginini, M. bovis, M. fermentans, M. gallinaceum, M. gallisepticum, M. synoviae, M. hominis, M. hyorhinis, M. orale, M. salivarium, and Acholeplasma laidlawii) known to be most frequently associated with contamination of cell substrates and cell lines in research laboratories or manufacturing facilities. The infection of MDCK cells with serial dilutions of each mycoplasma species demonstrated that these common cell line contaminants can be detected reliably after 7-day enrichment in MDCK cell culture at contamination levels of 0.05 to 0.25 CFU/ml. The High Five insect cell line was also found to be able to support the efficient growth of most mycoplasma species tested, except for M. hyorhinis strain DBS1050. However, mycoplasma growth in insect cell culture was demonstrated to be temperature dependent, and the most efficient growth was observed when the incubation temperature was increased from 28 degrees C to between 35 and 37 degrees C. We believe that this type of mycoplasma enrichment is one of the most promising approaches for improving the purity and safety testing of cell substrates and other cell-derived biologics and pharmaceuticals.

Hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus type-1 (HIV-1) are transfusion-transmitted human pathogens that have a major impact on blood safety and public health worldwide. We developed a microarray multiplex assay for the simultaneous detection and discrimination of these three viruses. The microarray consists of 16 oligonucleotide probes, immobilized on a silylated glass slide. Amplicons from multiplex PCR were labeled with Cy-5 and hybridized to the microarray. The assay detected 1 International Unit (IU), 10 IU, 20 IU of HBV, HCV, and HIV-1, respectively, in a single multiplex reaction. The assay also detected and discriminated the presence of two or three of these viruses in a single sample. Our data represent a proof-of-concept for the possible use of highly sensitive multiplex microarray assay to screen and confirm the presence of these viruses in blood donors and patients.

An oligonucleotide microarray hybridization method for identification of most known measles virus (MV) genotypes was developed. Like the conventional genotyping method, the microarray relied on detecting sequence differences in the 450-nucleotide region coding for the COOH-terminal 150 amino acids of the nucleoprotein (N). This region was amplified using PCR primers binding to all known MV genotypes. The microarray included 71 pairs of oligonucleotide probes (oligoprobes) immobilized on glass slides. Each pair consisted of a genotype-specific oligoprobe, which matched the sequence of only one target genotype, and a control oligoprobe, which contained mismatches at the nucleotide positions unique to this genotype. A pattern recognition algorithm based on cluster analysis of the ratios of hybridization signals from specific and control oligoprobes was used to identify the specific MV genotype. Following the initial validation, the method was used for rapid genotyping of two panels of coded samples. The results of this study showed good sensitivity (90.7%), specificity (100%), and genotype agreement (91.8%) for the new method compared to the results of genotyping conducted using phylogenetic analysis of viral sequences of the C terminus of the N gene. In addition, the microarray demonstrated the ability to identify potential new genotypes of MV based on the similarity of their hybridization patterns with those of known MV genotypes.

An oligonucleotide microarray for detection and identification of orthopoxviruses was developed. Genus specific and orthopoxvirus species-specific regions of the genes encoding chemokine binding and alpha/beta-interferon binding proteins were used as a target. The developed microarray allows the variola, monkeypox, cowpox, vaccinia, camel-pox and ectromelia (mousepox) viruses to be distinguished with a high degree of reliability.

To identify the hantaviruses causing hemorrhagic fever with renal syndrome (HFRS) in the Far East of Russia, blood samples collected from HFRS patients in 1994-1998, were examined by reverse transcription-polymerase chain reaction. In addition, 36 sera were tested by an immunofluorescence assay for antibodies against Hantaan, Seoul, Puumala, and Khabarovsk viruses, and 54 samples were tested by plaque reduction neutralization test. With both serological assays, the highest antibody titers were to Hantaan and/or Seoul viruses. Of 110 blood samples 36 were found RT-PCR positive. Phylogenetic analysis the sequences of a 256-nucleotide (nt) fragment of the hantavirus M genome segment revealed at least 3 genetically distinct hantavirus lineages. Nucleotide sequence comparison showed that two of the lineages, designated as FE and Amur (AMR), differed from one another by 15.9-21.2% and from Hantaan virus by 9.8-17.5%. The third lineage, VDV, differed from Seoul virus by 2.6-5.1%. All S segment sequences were from FE lineage, and differed from Hantaan virus by 10.7-12.6%. Thirty of the 36 (83%) analyzed sequences were found to be the FE genotype, which is very similar to that of Hantaan virus, strain 76-118. Of the remaining hantaviruses, 11% were the AMR genotype, and 6% the VDV genotype, which are genetically novel genotypes of Hantaan or Seoul viruses, respectively.

Alastrim variola minor virus, which causes mild smallpox, was first recognized in Florida and South America in the late 19th century. Genome linear double-stranded DNA sequences (186,986 bp) of the alastrim virus Garcia-1966, a laboratory reference strain from an outbreak associated with 0.8% case fatalities in Brazil in 1966, were determined except for a 530-bp fragment of hairpin-loop sequences at each terminus. The DNA sequences (EMBL Accession No. Y16780) showed 206 potential open reading frames for proteins containing >/=60 amino acids. The amino acid sequences of the putative proteins were compared with those reported for vaccinia virus strain Copenhagen and the Asian variola major strains India-1967 and Bangladesh-1975. About one-third of the alastrim viral proteins were 100% identical to correlates in the variola major strains and the remainder were >/=95% identical. Compared with variola major virus DNA, alastrim virus DNA has additional segments of 898 and 627 bp, respectively, within the left and right terminal regions. The former segment aligns well with sequences in other orthopoxviruses, particularly cowpox and vaccinia viruses, and the latter is apparently alastrim-specific., (Copyright 2000 Academic Press.)

The enzymes of the FauI restriction-modification system from the Flavobacterium aquatile strain recognize the non-palindromic sequence 5'-CCCGC-3'/3'-GG-GCG-5'. We have cloned the gene encoding the DNA modifying component of this system and determined its nucleotide sequence. The deduced amino acid sequence contains ten conserved motifs characteristic for [cytosine-5] DNA methyltransferases. Part of the gene sequence that encodes the putative target recognizing domain of the M.FauI shows some homology with the downstream region, thus indicating that duplication of the DNA segment was probably involved in the gene evolution.

Genome DNA terminal region sequences were determined for a Brazilian alastrim variola minor virus strain Garcia-1966 that was associated with an 0.8% case-fatality rate and African smallpox strains Congo-1970 and Somalia-1977 associated with variola major (9.6%) and minor (0.4%) mortality rates, respectively. A base sequence identity of > or = 98.8% was determined after aligning 30 kb of the left- or right-end region sequences with cognate sequences previously determined for Asian variola major strains India-1967 (31% death rate) and Bangladesh-1975 (18.5% death rate). The deduced amino acid sequences of putative proteins of > or = 65 amino acids also showed relatively high identity, although the Asian and African viruses were clearly more related to each other than to alastrim virus. Alastrim virus contained only 10 of 70 proteins that were 100% identical to homologs in Asian strains, and 7 alastrim-specific proteins were noted.

This study reports completion of the genetic characterization of the entire genome of Sin Nombre (SN) virus (NMH10) detected in autopsy tissues from a patient who died of hantavirus pulmonary syndrome (HPS). The large (L) genome segment was found to be 6,562 nucleotides in length and encoded a putative L polymerase that was 2,153 amino acids in length. No evidence of segment reassortment with other well-characterized hantaviruses was obtained. The sequence of the entire S, M, and L genome segments of SN virus (strain NMR11) isolated from a mouse (trapped in the residence of the patient infected with SN virus [NMH10]) by passage two times in Peromyscus maniculatus and then by five passages in E6 Vero cells was determined and compared with that of the virus detected in autopsy tissues. Only 16 nucleotide differences were detected between the virus genomes, and none of these resulted in virus protein amino acid substitutions. Determination of the exact 5'- and 3'-terminal sequences of all genome segments of SN virus and representatives of other serologic groups in the Hantavirus genus, family Bunyaviridae, showed the existence of conserved nucleotide domains that may be involved in important regulatory mechanisms, such as RNA encapsidation, polymerase binding, and control of transcription and replication.

Sequencing and computer analysis of the variola major virus strain India-1967 (VAR-IND) genome segment (53,018 bp) from the right terminal region have been carried out. Fifty nine potential open reading frames (ORFs) of over 60 amino acid residues have been identified. Structure-function organization of VAR-IND DNA segment under study was compared with the previously reported sequences from the analogous genomic regions of vaccinia virus strains Copenhagen (VAC-COP) and Western Reserve (VAC-WR) and variola virus strain Harvey (VAR-HAR). Multiple distinctions in the genetic map of VAR-IND from VAC-COP and VAC-WR have been revealed along with the high similarity to the corresponding VAR-HAR segment. Possible functions of the predicted viral proteins and the effect of their differences on the features of orthopoxviruses are discussed.

A detection technique for hepatitis A virus (HAV) RNA by means of molecular hybridization using ss-biotinated DNA probe on the basis of M13 bacteriophage is described. The technique sensitivity reached 1-10 pg of control DNA or 100-500 pg of HAV. The experiments for detection of HAV carriers among the patients and contacts from foci of HAV outbreaks were carried out. A comparative analysis of the above technique and enzyme immunoassay and amplification technique was done and good coincidence of the results was demonstrated.

Computer analysis of variola major virus (VAR) genomic fragment bounded by open reading frames (ORFs) D1R and A33L which is 47,961 bp long revealed 46 potential ORFs. The VAR proteins were compared with the analogous proteins of vaccinia virus strain Copenhagen. The subunits of DNA-dependent RNA polymerase, as well as the transcription factors, mRNA capping enzymes, and proteins necessary for the virion morphogenesis proved to be highly conservative within orthopoxviruses. The most pronounced differences between the VAR genome fragment under study and the corresponding vaccinia virus fragment were revealed in the vicinity of the gene encoding the A-type inclusion body protein. The possible functions of the analyzed viral proteins are discussed.

Sequencing of variola virus (VAR) genome region of 43069 bp was carried out. This area contains 42 potential genes. Computer analysis of proteins coding for these viral genes was done. We compared VAR proteins with the those of vaccinia virus. The region studied is conservative for orthopoxviruses.

The nucleotide sequence of the 3913 base pair XhoI O fragment located in an evolutionary variable region adjacent to the right end of the genome of ectromelia virus (EMV) was determined. The sequence contains two long open reading frames coding for putative proteins of 559 amino acid residues (p65) and 344 amino acid residues (p39). Amino acid database searches showed that p39 is closely related to vaccinia virus (VV), strain WR, B22R gene product (C12L gene product of strain Copenhagen), which belongs to the family of serine protease inhibitors (serpins). Despite the overall high conservation, differences were observed in the sequences of p39, B22R, and C12L in the site known to interact with proteases in other serpins, suggesting that the serpins of EMV and two strains of VV may all inhibit proteases with different specificities. The gene coding for the ortholog of p65 is lacking in the Copenhagen strain of vaccinia virus; the WR strain contains a truncated variant of this gene (B21R) potentially coding for a small protein (p16) corresponding to the C-terminal region of p65. p65 is a new member of the family of poxvirus proteins including vaccinia virus proteins A55R, C2L and F3L, and a group of related proteins of leporipoxviruses, Shope fibroma and myxoma viruses (T6, T8, T9, M9). These proteins are homologous to the Drosophila protein Kelch involved in egg development. Both Kelch protein and the related poxvirus proteins contain two distinct domains. The N-terminal domain is related to the similarly located domains of transcription factors Ttk, Br-C (Drosophila), and KUP (human), and GCL protein involved in early development in Drosophila. The C-terminal domain consists of an array of four to five imperfect repeats and is related to human placental protein MIPP. Phylogenetic analysis of the family of poxvirus proteins showed that their genes have undergone a complex succession of duplications, and complete or partial deletions.

A fast-growing cytopathic isolate of human hepatitis A virus (strain MB-7) was derived from fecal samples of infected patients and adapted to growth in FRhK-4 cell culture. A positive serum standard against HAV and electron microscopy were used to demonstrate that MB-7 belonged to human hepatitis A virus. The strain MB-7 induced plaque formation in FRhK-4 under agar overlay after 10-12 days of incubation. The PCR products of gene VP1 were cloned in E. coli and its primary structure was determined. MB-7 was shown to have more homology with HAV strains isolated in the USA and China.

DNA of the variola major virus strain India-1967 in the region of HindIII M, L, I fragments has been sequenced. Analysis of this sequence of 18029 bp revealed 19 potential open reading frames (ORFs). Four proposed proteins (L2R, H9R, L5L, L6R) contain metal-binding domains. Comparison of the variola virus (VAR) and vaccinia virus strain Copenhagen (COP) sequences show that the main differences are between proteins L1R and I5R. L1R contains 6 additional amino acid residues on the C-terminus. The protein I5R of VAR contains three Ca2+ binding domains but this COP has deletions in 2 of the 3 established domains. Possible functions of the predicted viral polypeptides are discussed.

HindIII and XhoI genome fragments of variola major virus strain India-1967 were inserted into the bacterial plasmids and cosmid. Sequencing and computer analysis of the region of HindIII M, L, and I DNA fragments of the virus studied have been carried out.

The method for analysis of microorganisms for the presence of the modification-restriction systems has been developed. The method has permitted to detect more than 10 new producing strains of restrictases including microorganisms of Rhizobium genus. Some of them are promising for practical use. It has been shown that using selection of clones the strain productivity can be increased. The purification process for the majority of restrictases has been proposed. Some physical and catalytic properties of new enzymes have been studied.

Two genetics markers: the herpes simplex virus thymidine kinase and Escherichia coli beta-galactosidase genes were inserted into the 36K protein gene of vaccinia virus located in a HindIII-P DNA fragment. An unstability of recombinant viruses with Lac(+)-phenotype were discovered. A mechanism of viruses unstable variants formation was proposed, it was confirmed by the results of hybridisation analyses of virus recombinant genomes. The importance of a late nonstructural 36K protein gene for virus reproduction was demonstrated.

For preparing a DNA fragment with unique protruding ends, plasmid vectors pMB123 and pMB124 were constructed by inserting a synthetic polylinker into plasmid pUR222 at the EcoRI-PstI sites. The polylinker contains two FokI and HgaI sites at its ends in opposite orientation flanking a combination of SalGI, AccI, HindII, HindIII (the latter site is absent from pMB124) and BamHI sites. DNA fragment cloned at the SalGI and BamHI sites can be regenerated by either FokI or HgaI treatment, the SalGI and BamHI sites being deleted from the cloned sequence. Fragments coding for parts of human interleukin-2 were cloned in these vectors.

Reverse transcription afforded DNA-copies of the 3'-terminal sequence of mouse encephalomyocarditis virus RNA. Cloning of this cDNA in pBR 322 plasmid and sequencing of DNA of one of the clones established the structure (552 nucleotide residues) of the 3'-terminus of viral RNA. This region contains a non-translatable fragment (126 nucleotides) and also the fragment coding for the C-terminus of viral replicase.

Reverse transcription produced DNA-copies of the 3'-terminal sequence of mouse encephalomyocarditis virus RNA. Cloning and sequencing of this cDNA afforded the structure of the 3'-terminus of viral RNA over the length of 2988 nucleotide residues. The probable sites of proteolytic cleavage of the immature protein have been determined and the homology with the respective poliovirus proteins has been established.

The genome of hepatitis A virus (HAV) isolated from spontaneously infected African vervet monkey (Cercopithecus aethiops) has been cloned and partially sequenced. Comparison of genome fragments (1248 and 162 bp) from the 3D (RNA polymerase) region with the corresponding parts of human HAV genomes revealed a high degree of heterogeneity: there were altogether 257 nucleotide changes leading to 44 substitutions in predicted amino acid sequence, i.e. 89% amino acid identity. This divergence is considered to be significantly greater than genomic variations usually found among human HAV strains, where amino acid identity in the 3D region is over 98%.

Photoreactive derivatives of tRNAPhe (E. coli) were synthesized by alkylation of the tRNAPhe with 4-(N-2-chloroethyl-N-methylamino)benzylamine and subsequent treatment with 1.4--dinitro-5-fluorophenylazide. The derivatives are active in binding with ribosomes and phenylalanyl-tRNA synthetase when the extent of modification is lower than 3 reactive groups per tRNAPhe molecule. Under irradiation the derivatives modify exclusively the beta-subunit of the phenylalanyl-tRNA synthetase.

The recognition sequence and cleavage point of restriction endonuclease VspI have been determined as 5'-AT decreases TAAT. This enzyme is not isoschizomer of any known restriction endonucleases. DNA pBR322 contains a single VspI recognition sequence in position 3539. Therefore this enzyme may be used for cloning DNA in the VspI site in AmpR-gene of pBR322.