Publications by authors named "Paola Pilo"

44 Publications

Prevalence and phylogeny of Chlamydiae and hemotropic mycoplasma species in captive and free-living bats.

BMC Microbiol 2020 06 26;20(1):182. Epub 2020 Jun 26.

Institute of Veterinary Pathology, Vetsuisse Faculty, University of Zurich, Zurich, Switzerland.

Background: Bats are hosts for a variety of microorganisms, however, little is known about the presence of Chlamydiales and hemotropic mycoplasmas. This study investigated 475 captive and free-living bats from Switzerland, Germany, and Costa Rica for Chlamydiales and hemotropic mycoplasmas by PCR to determine the prevalence and phylogeny of these organisms.

Results: Screening for Chlamydiales resulted in a total prevalence of 31.4%. Positive samples originated from captive and free-living bats from all three countries. Sequencing of 15 samples allowed the detection of two phylogenetically distinct groups. These groups share sequence identities to Chlamydiaceae, and to Chlamydia-like organisms including Rhabdochlamydiaceae and unclassified Chlamydiales from environmental samples, respectively. PCR analysis for the presence of hemotropic mycoplasmas resulted in a total prevalence of 0.7%, comprising free-living bats from Germany and Costa Rica. Phylogenetic analysis revealed three sequences related to other unidentified mycoplasmas found in vampire bats and Chilean bats.

Conclusions: Bats can harbor Chlamydiales and hemotropic mycoplasmas and the newly described sequences in this study indicate that the diversity of these bacteria in bats is much larger than previously thought. Both, Chlamydiales and hemotropic mycoplasmas are not restricted to certain bat species or countries and captive and free-living bats can be colonized. In conclusion, bats represent another potential host or vector for novel, previously unidentified, Chlamydiales and hemotropic mycoplasmas.
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http://dx.doi.org/10.1186/s12866-020-01872-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7318495PMC
June 2020

Comparison of Two Multilocus Sequence Typing Schemes for Mycoplasma bovis and Revision of the PubMLST Reference Method.

J Clin Microbiol 2020 05 26;58(6). Epub 2020 May 26.

Infectious Bacterial Diseases Research Unit, USDA/Agricultural Research Service/National Animal Disease Center, Ames, Iowa, USA.

causes pneumonia, pharyngitis, otitis, arthritis, mastitis, and reproductive disorders in cattle and bison. Two multilocus sequence typing (MLST) schemes have been developed for , with one serving as the PubMLST reference method, but no comparison of the schemes has been undertaken. Although the PubMLST scheme has proven to be highly discriminatory and informative, the recent discovery of isolates missing one of the typing loci, , raises concern about its suitability for continued use. The goal of our study was to compare the performance of the two MLST schemes and identify a new reference scheme capable of fully typing all isolates. We evaluated 448 isolates from diverse geographic and anatomic sites that collectively represent cattle, bison, deer, and a goat. The discrimination indexes (DIs) for the PubMLST and the alternative scheme are 0.909 (91 sequence types [STs]) and 0.842 (77 STs), respectively. Although the PubMLST scheme outperformed the alternative scheme, the locus must be retired from the PubMLST scheme if it is to be retained as a reference method. The DI obtained using the six remaining PubMLST loci (0.897, 79 STs) fails to reach the benchmark recommended for a reference method (0.900), mandating the addition of a seventh locus. Comparative analysis of genome sequences from the isolates used here identified the locus from the alternative scheme as the optimal replacement for This revised scheme, which will be implemented as the new PubMLST reference method, has a DI of 0.914 and distinguishes 88 STs from the 448 isolates evaluated.
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http://dx.doi.org/10.1128/JCM.00283-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7269390PMC
May 2020

Large-Scale Analysis of the Genome Identified Non-essential, Adhesion- and Virulence-Related Genes.

Front Microbiol 2019 13;10:2085. Epub 2019 Sep 13.

Department of Infectious Diseases and Pathobiology, Vetsuisse Faculty, Institute of Veterinary Bacteriology, University of Bern, Bern, Switzerland.

is an important pathogen of cattle causing bovine mycoplasmosis. Clinical manifestations are numerous, but pneumonia, mastitis, and arthritis cases are mainly reported. Currently, no efficient vaccine is available and antibiotic treatments are not always satisfactory. The design of new, efficient prophylactic and therapeutic approaches requires a better understanding of the molecular mechanisms responsible for pathogenicity. Random transposon mutagenesis has been widely used in species to identify potential gene functions. Such an approach can also be used to screen genomes and search for essential and non-essential genes for growth. Here, we generated a random transposon mutant library of strain JF4278 containing approximately 4000 independent insertion sites. We then coupled high-throughput screening of this mutant library to transposon sequencing and bioinformatic analysis to identify non-essential, adhesion- and virulence-related genes. Three hundred and fifty-two genes of were assigned as essential for growth in rich medium. Among the remaining non-essential genes, putative virulence-related factors were subsequently identified. The complete mutant library was screened for adhesion using primary bovine mammary gland epithelial cells. Data from this assay resulted in a list of conditional-essential genes with putative adhesion-related functions by identifying non-essential genes for growth that are essential for host cell-adhesion. By individually assessing the adhesion capacity of six selected mutants, two previously unknown factors and the adhesin TrmFO were associated with a reduced adhesion phenotype. Overall, our study (i) uncovers new, putative virulence-related genes; (ii) offers a list of putative adhesion-related factors; and (iii) provides valuable information for vaccine design and for exploring biology, pathogenesis, and host-interaction.
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http://dx.doi.org/10.3389/fmicb.2019.02085DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6753880PMC
September 2019

Coinfection of Swiss cattle with bovine parainfluenza virus 3 and at acute and chronic stages of bovine respiratory disease complex.

J Vet Diagn Invest 2019 Sep 27;31(5):674-680. Epub 2019 Jun 27.

Institute of Animal Pathology (Mehinagic, Stokar-Regenscheit), Department of Infectious Diseases and Pathobiology.

Viral agents such as bovine respiratory syncytial virus (BRSV) and bovine parainfluenza virus 3 (BPIV-3) are considered primary infectious agents in bovine respiratory disease complex (BRDC). Information regarding the pathogenesis of BRDC is scarce, especially at an advanced chronicity stage, in addition to ongoing coinfection with other primary agents such as . Based on a retrospective review of histology slides from 104 autopsy cases, we classified cases according to type of pneumonia and chronicity. We performed immunohistochemistry (IHC) for BRSV, BPIV-3, and as well as real-time PCR (rtPCR) for on lung tissue of all 104 cases and correlated results with the morphologic type of pneumonia. Histomorphologically, 79 cases were classified as bronchopneumonia, 16 as bronchointerstitial pneumonia, and 9 as interstitial pneumonia. In 89 cases, at least 1 of the investigated agents was detected by IHC; 44 of these cases had a coinfection. BPIV-3 was the predominant agent present, as a single infection in 39 cases, and in coinfection with in 39 cases. Comparing the detection methods for , rtPCR was more specific and sensitive than IHC. The combination of both methods provided a good visual tool for assessing severity and distribution of antigen within the tissue. Unlike BRSV, BPIV-3 and persisted in chronic BRDC, suggesting ongoing impairment of defense mechanisms in the lung.
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http://dx.doi.org/10.1177/1040638719861686DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6727125PMC
September 2019

Systemic infection in European perch with thermoadapted virulent Aeromonas salmonicida (Perca fluviatilis).

J Fish Dis 2019 May 26;42(5):685-691. Epub 2019 Feb 26.

Centre for Fish and Wildlife Health, University of Bern, Bern, Switzerland.

In non-salmonid fish, Aeromonas salmonicidacan cause local infections with severe skin ulcerations, known as atypical furunculosis. In this study, we present a systemic infection by a virulent A. salmonicidain European perch (Perca fluviatilis).This infection was diagnosed in a Swiss warm water recirculation aquaculture system. The isolate of A.  salmonicida encodes a type three secretion system (TTSS) most likely located on a plasmid similar to pAsa5/pASvirA, which is known to specify one of the main virulence attributes of the species A. salmonicida. However, the genes specifying the TTSS of the perch isolate show a higher temperature tolerance than strains isolated from cold-water fish. The function of the TTSS in virulence was verified in a cytotoxicity test using bluegill fry and epithelioma papulosum cyprinid cells.
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http://dx.doi.org/10.1111/jfd.12970DOI Listing
May 2019

Bovine Epithelial Infection Models for .

Front Cell Infect Microbiol 2018 18;8:329. Epub 2018 Sep 18.

Department of Infectious Diseases and Pathobiology, Vetsuisse Faculty, Institute of Veterinary Bacteriology University of Bern, Bern, Switzerland.

causes bovine mycoplasmosis. The major clinical manifestations are pneumonia and mastitis. Recently an increase in the severity of mastitis cases was reported in Switzerland. At the molecular level, there is limited understanding of the mechanisms of pathogenicity of . Host-pathogen interactions were primarily studied using primary bovine blood cells. Therefore, little is known about the impact of on other cell types present in infected tissues. Clear phenotypes linked to the virulence of strains or tissue predilection of specific strains have not yet been described. We adapted bovine systems to investigate infection of epithelial cells with using a cell line (MDBK: Madin-Darby bovine kidney cells) and two primary cells (PECT: bovine embryonic turbinate cells and bMec: bovine mammary gland epithelial cells). Two strains isolated before and after the emergence of severe mastitis cases were selected. Strain JF4278 isolated from a cow with mastitis and pneumonia in 2008 and strain L22/93 isolated in 1993 were used to assess the virulence of genotypes toward epithelial cells with particular emphasis on mammary gland cells. Our findings indicate that is able to adhere to and invade different epithelial cell types. Higher titers of JF4278 than L22/93 were observed in co-cultures with cells. The differences in titers reached between the two strains was more prominent for bMec cells than for MDBK and PECT cells. Moreover, strain L22/93 induced apoptosis in MDBK cells and cytotoxicity in PECT cells but not in bMec cells. Dose-dependent variations in proliferation of primary epithelial cells were observed after infection. Nevertheless, an indisputable phenotype that could be related to the increased virulence toward mammary gland cells is not obvious.
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http://dx.doi.org/10.3389/fcimb.2018.00329DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6153342PMC
September 2019

Phylogenetic Lineages of in Animals.

Authors:
Paola Pilo

Front Cell Infect Microbiol 2018 31;8:258. Epub 2018 Jul 31.

Vetsuisse Faculty, Institute of Veterinary Bacteriology, Department of Infectious Diseases and Pathobiology, University of Bern, Bern, Switzerland.

Tularemia is a zoonotic disease caused by the facultative intracellular bacterium . This microorganism can infect a plethora of animal species and its ecology is particularly complex. Much research was performed to understand its biology but many questions are still open, especially concerning the life cycle of this bacterium in the environment related to physical and biological parameters. Numerous animals are major hosts of but precise reservoir species are not yet well defined. Moreover, the exact range of species susceptible to tularemia is not clear and is complicated by the differences in virulence and ecology observed among the subspecies of . Indeed, different life cycles in nature, including the animal species concerned, were previously described for subsp. and subsp. . Recently, molecular techniques showing adequate discrimination between strains were developed, leading to the possibility to investigate links between phylogenetic lineages and infection in animals. New perspectives in research are now possible thanks to the information available and the simplicity of the molecular procedures. Current studies are unfolding the evolution of and these developments will lead to the elucidation of geographical and ecological differences observed by veterinarians, microbiologists and conservation biologists. However, systematic, coordinated collection of data and extensive sampling are important to efficiently assemble the findings of future research.
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http://dx.doi.org/10.3389/fcimb.2018.00258DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6079424PMC
August 2019

Pathogenicity, population genetics and dissemination of Bacillus anthracis.

Infect Genet Evol 2018 10 20;64:115-125. Epub 2018 Jun 20.

Dean's Office, Vetsuisse Faculty, University of Bern, Bern, Switzerland. Electronic address:

Bacillus anthracis, the etiological agent of anthrax, procures its particular virulence by a capsule and two AB type toxins: the lethal factor LF and the edema factor EF. These toxins primarily disable immune cells. Both toxins are translocated to the host cell by the adhesin-internalin subunit called protective antigen PA. PA enables LF to reach intra-luminal vesicles, where it remains active for long periods. Subsequently, LF translocates to non-infected cells, leading to inefficient late therapy of anthrax. B. anthracis undergoes slow evolution because it alternates between vegetative and long spore phases. Full genome sequence analysis of a large number of worldwide strains resulted in a robust evolutionary reconstruction of this bacterium, showing that B. anthracis is split in three main clades: A, B and C. Clade A efficiently disseminated worldwide underpinned by human activities including heavy intercontinental trade of goat and sheep hair. Subclade A.Br.WNA, which is widespread in the Northern American continent, is estimated to have split from clade A reaching the Northern American continent in the late Pleistocene epoch via the former Bering Land Bridge and further spread from Northwest southwards. An alternative hypothesis is that subclade A.Br.WNA. evolved from clade A.Br.TEA tracing it back to strains from Northern France that were assumingly dispatched by European explorers that settled along the St. Lawrence River. Clade B established mostly in Europe along the alpine axis where it evolved in association with local cattle breeds and hence displays specific geographic subclusters. Sequencing technologies are also used for forensic applications to trace unintended or criminal acts of release of B. anthracis. Under natural conditions, B. anthracis generally affects domesticated and wild ruminants in arid ecosystems. The more recently discovered B. cereus biovar anthracis spreads in tropical forests, where it threatens particularly endangered primate populations.
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http://dx.doi.org/10.1016/j.meegid.2018.06.024DOI Listing
October 2018

Population Genomics of subsp. and its Implication on the Eco-Epidemiology of Tularemia in Switzerland.

Front Cell Infect Microbiol 2018 22;8:89. Epub 2018 Mar 22.

Spiez Laboratory, Federal Office for Civil Protection, Spiez, Switzerland.

Whole genome sequencing (WGS) methods provide new possibilities in the field of molecular epidemiology. This is particularly true for monomorphic organisms where the discriminatory power of traditional methods (e.g., restriction enzyme length polymorphism typing, multi locus sequence typing etc.) is inadequate to elucidate complex disease transmission patterns, as well as resolving the phylogeny at high resolution on a micro-geographic scale. In this study, we present insights into the population structure of subsp. , the causative agent of tularemia in Switzerland. A total of 59 isolates were obtained from castor bean ticks (, animals and humans and a high resolution phylogeny was inferred using WGS methods. The majority of the population in Switzerland belongs to the west European B.11 clade and shows an extraordinary genetic diversity underlining the old evolutionary history of the pathogen in the alpine region. Moreover, a new B.11 subclade was identified which was not described so far. The combined analysis of the epidemiological data of human tularemia cases with the whole genome sequences of the 59 isolates provide evidence that ticks play a pivotal role in transmitting to humans and other vertebrates in Switzerland. This is further underlined by the correlation of disease risk estimates with climatic and ecological factors influencing the survival of ticks.
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http://dx.doi.org/10.3389/fcimb.2018.00089DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5875085PMC
April 2019

Sensitive and Specific Recombinase Polymerase Amplification Assays for Fast Screening, Detection, and Identification of Bacillus anthracis in a Field Setting.

Appl Environ Microbiol 2018 06 17;84(11). Epub 2018 May 17.

Centre de Technologies Moléculaires Appliquées, Institut de Recherche Expérimentale et Clinique, Université catholique de Louvain, Brussels, Belgium.

Four isothermal recombinase polymerase amplification (RPA) assays were developed for fast in-field identification of The RPA assays targeted three specific sequences (i.e., the BA_5345 chromosomal marker, the lethal factor [from pXO1], and the capsule-biosynthesis-related [from pXO2]) and a conserved sequence in the adenylate cyclase gene () for the group. -specific RPA assays were tested first with purified genomic DNAs ( = 60), including 11 representatives of , and then with soil ( = 8) and white powder ( = 8) samples spiked with inactivated spores and/or other biological agents. The RPA assays were also tested in another laboratory facility, which blindly provided DNA and lysate samples ( = 30, including 20 strains). RPA assays displayed 100% specificity and sensitivity. The hands-off turnaround times at 42°C ranged from 5 to 6 min for 10 genomic copies. The analytical sensitivity of each RPA assay was ∼10 molecules per reaction. In addition, the BA_5345 and RPA assays were assessed under field conditions with a series of surface swabs ( = 13, including 11 swabs contaminated with spores) that were blindly brought to the field laboratory by a chemical, biological, radiological, and nuclear (CBRN) sampling team. None of the 13 samples, except the control, tested positive for , and all samples that had been harvested from spore-contaminated surfaces tested positive with the RPA assay. All three -specific RPA assays proved suitable for rapid and reliable identification of and therefore could easily be used by first responders under field conditions to quickly discriminate between a deliberate release of spores and a hoax attack involving white powder. In recent decades, particularly following the 11 September 2001 and Amerithrax attacks, the world has experienced attempts to sow panic and chaos in society through thousands of white-powder copycats using household powders to mimic real bioterrorism attacks. In such circumstances, field-deployable detection methods are particularly needed to screen samples collected from the scene. The aim is to test the samples directly using a fast and reliable assay for detection of the presence of While this would not preclude further confirmatory tests from being performed in reference laboratories, it would bring useful, timely, and relevant information to local crisis managers and help them make appropriate decisions without having to wait for quantitative PCR results (with turnaround times of a few hours) or phenotypic identification and sequencing (with turnaround times of a few days). In the current investigation, we developed a set of isothermal RPA assays for the rapid screening and identification of in powders and soil samples, with the purpose of discriminating a deliberate release of spores from a hoax attack involving white powder; this would also apply to dispersion by spraying of aerosolized forms of Further work is now ongoing to confirm the first observations and validate the on-site use of these assays by first responders.
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http://dx.doi.org/10.1128/AEM.00506-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5960963PMC
June 2018

Mycoplasma bovis co-infection with bovine viral diarrhea virus in bovine macrophages.

Vet Res 2018 01 9;49(1). Epub 2018 Jan 9.

Institute of Veterinary Bacteriology, Department of Infectious Diseases and Pathobiology, Vetsuisse Faculty, University of Bern, Laenggass-Str. 122, 3001, Bern, Switzerland.

Several studies suggest that synergisms between Mycoplasma bovis and other microorganisms might exacerbate disease outcome of bovine mycoplasmosis. Screening several bovine cell types to assess their potential use as in vitro infection models for M. bovis, it was observed that a widely used cell line of bovine macrophages (Bomac cells) is in fact persistently infected with bovine viral diarrhea virus (BVDV). The cell line was first cured of this virus allowing comparative studies between both cell lines. Subsequently, uptake and co-culture of two M. bovis strains of different clonal complexes with Bomac cells contaminated with BVDV and in BVDV-free Bomac cells were assessed. Additionally, cell viability, cytotoxicity and induction of apoptosis after infection with M. bovis were evaluated. No differences in the levels of uptake and growth in co-culture were observed between the two Bomac cell types and both M. bovis strains. Cytotoxicity was increased after infection of BVDV-free cells with one of the two strains, while apoptotic cell death was slightly induced by this strain in both cell lines. Overall, the presence or absence of BVDV in Bomac cells did not grossly change the parameters tested upon infection with M. bovis. Nevertheless, this cell model is very useful when studying viral co-infections with bacteria and could also be used for multiple co-infections. Considering the broad contamination of cell cultures with BVDV, careful screening for this virus should routinely be performed as its presence might be relevant depending on the molecular mechanisms being investigated.
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http://dx.doi.org/10.1186/s13567-017-0499-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5761114PMC
January 2018

Long-range dispersal moved into Western Europe from the East.

Microb Genom 2016 12 12;2(12):e000100. Epub 2016 Dec 12.

1​Department of Clinical Microbiology and the Laboratory for Molecular Infection Medicine Sweden, Umeå University, Umeå, Sweden.

For many infections transmitting to humans from reservoirs in nature, disease dispersal patterns over space and time are largely unknown. Here, a reversed genomics approach helped us understand disease dispersal and yielded insight into evolution and biological properties of , the bacterium causing tularemia. We whole-genome sequenced 67 strains and characterized by single-nucleotide polymorphism assays 138 strains, collected from individuals infected 1947-2012 across Western Europe. We used the data for phylogenetic, population genetic and geographical network analyses. All strains (=205) belonged to a monophyletic population of recent ancestry not found outside Western Europe. Most strains (=195) throughout the study area were assigned to a star-like phylogenetic pattern indicating that colonization of Western Europe occurred via clonal expansion. In the East of the study area, strains were more diverse, consistent with a founder population spreading from east to west. The relationship of genetic and geographic distance within the population was complex and indicated multiple long-distance dispersal events. Mutation rate estimates based on year of isolation indicated null rates; in outbreak hotspots only, there was a rate of 0.4 mutations/genome/year. Patterns of nucleotide substitution showed marked AT mutational bias suggestive of genetic drift. These results demonstrate that tularemia has moved from east to west in Europe and that has a biology characterized by long-range geographical dispersal events and mostly slow, but variable, replication rates. The results indicate that mutation-driven evolution, a resting survival phase, genetic drift and long-distance geographical dispersal events have interacted to generate genetic diversity within this species.
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http://dx.doi.org/10.1099/mgen.0.000100DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5359409PMC
December 2016

A dominant lineage of Mycoplasma bovis is associated with an increased number of severe mastitis cases in cattle.

Vet Microbiol 2016 Nov 11;196:63-66. Epub 2016 Oct 11.

Institute of Veterinary Bacteriology, Vetsuisse Faculty, University of Bern, Laenggassstrasse 122, 3012, Bern, Switzerland. Electronic address:

Mycoplasma bovis is the most frequent etiologic agent of bovine mycoplasmosis. It causes various diseases in bovines and considerable economic loss due to the lack of effective treatment or preventive measures such as vaccination. In contrast to the US, where M. bovis-mastitis has been reported for a long time, M. bovis infections in Switzerland and Austria were predominantly associated with pneumonia and subclinical mastitis. However, since 2007 the situation has changed with the emergence of severe M. bovis-associated mastitis cases in both countries. In order to evaluate the molecular epidemiology of the bacteria isolated from these infections, recent and old Swiss, along with recent Austrian M. bovis isolates were analyzed by a typing method displaying intermediate resolution of evolutionary relationships among isolates called Multi-Locus Sequence Typing (MLST). The analysis of Swiss and Austrian M. bovis isolates revealed two major lineages. Isolates collected since 2007 in both countries cluster in the lineage I including ST5, ST33, ST34, 36, and ST38-40 (clonal complex 1), while all Swiss isolates recovered before 2007 cluster in the lineage II comprising ST17 and ST35 (clonal complex 5). Further investigations are necessary to understand if lineage I has a higher predilection or virulence toward mammary gland cells than the old lineage or if other factors are involved in the increased number of severe mastitis cases.
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http://dx.doi.org/10.1016/j.vetmic.2016.10.016DOI Listing
November 2016

Improving Exploitation of Whole Genome Sequencing Data for Public Health, Forensic Microbiology and Biosafety.

Authors:
Paola Pilo

EBioMedicine 2015 Nov 10;2(11):1566-7. Epub 2015 Nov 10.

Institute of Veterinary Bacteriology, Vetsuisse Faculty, University of Bern, Laenggassstrasse 122, 3012, Bern, Switzerland.

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http://dx.doi.org/10.1016/j.ebiom.2015.11.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4740334PMC
November 2015

Long-Lasting Fever and Lymphadenitis: Think about F. tularensis.

Case Rep Med 2015 3;2015:191406. Epub 2015 Nov 3.

Unit of Infectious Diseases, Department of Internal Medicine, HFR-Hôpital cantonal, 1708 Fribourg, Switzerland.

We report the case of glandular tularemia that developed in a man supposedly infected by a tick bite in Western Switzerland. Francisella tularensis (F. tularensis) was identified. In Europe tularemia most commonly manifests itself as ulcero-glandular or glandular disease; the diagnosis of tularemia may be delayed in glandular form where skin or mucous lesion is absent, particularly in areas which are assumed to have a low incidence of the disease.
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http://dx.doi.org/10.1155/2015/191406DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4646983PMC
November 2015

Dynamics of a Tularemia Outbreak in a Closely Monitored Free-Roaming Population of Wild House Mice.

PLoS One 2015 4;10(11):e0141103. Epub 2015 Nov 4.

Institute of Evolutionary Biology and Environmental Studies, University of Zurich, Zurich, Switzerland.

Infectious disease outbreaks can be devastating because of their sudden occurrence, as well as the complexity of monitoring and controlling them. Outbreaks in wildlife are even more challenging to observe and describe, especially when small animals or secretive species are involved. Modeling such infectious disease events is relevant to investigating their dynamics and is critical for decision makers to accomplish outbreak management. Tularemia, caused by the bacterium Francisella tularensis, is a potentially lethal zoonosis. Of the few animal outbreaks that have been reported in the literature, only those affecting zoo animals have been closely monitored. Here, we report the first estimation of the basic reproduction number R0 of an outbreak in wildlife caused by F. tularensis using quantitative modeling based on a susceptible-infected-recovered framework. We applied that model to data collected during an extensive investigation of an outbreak of tularemia caused by F. tularensis subsp. holarctica (also designated as type B) in a closely monitored, free-roaming house mouse (Mus musculus domesticus) population in Switzerland. Based on our model and assumptions, the best estimated basic reproduction number R0 of the current outbreak is 1.33. Our results suggest that tularemia can cause severe outbreaks in small rodents. We also concluded that the outbreak self-exhausted in approximately three months without administrating antibiotics.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0141103PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4633114PMC
June 2016

A Genomic Approach to Unravel Host-Pathogen Interaction in Chelonians: The Example of Testudinid Herpesvirus 3.

PLoS One 2015 5;10(8):e0134897. Epub 2015 Aug 5.

Department of Small Animal Clinical Sciences, College of Veterinary Medicine, University of Florida, Gainesville, Florida, United States of America.

We report the first de novo sequence assembly and analysis of the genome of Testudinid herpesvirus 3 (TeHV3), one of the most pathogenic chelonian herpesviruses. The genome of TeHV3 is at least 150,080 nucleotides long, is arranged in a type D configuration and comprises at least 102 open reading frames extensively co-linear with those of Human herpesvirus 1. Consistently, the phylogenetic analysis positions TeHV3 among the Alphaherpesvirinae, closely associated with Chelonid herpesvirus 5, a Scutavirus. To date, there has been limited genetic characterization of TeHVs and a resolution beyond the genotype was not feasible because of the lack of informative DNA sequences. To exemplify the potential benefits of the novel genomic information provided by this first whole genome analysis, we selected the glycoprotein B (gB) gene, for detailed comparison among different TeHV3 isolates. The rationale for selecting gB is that it encodes for a well-conserved protein among herpesviruses but is coupled with a relevant antigenicity and is consequently prone to accumulate single nucleotide polymorphisms. These features were considered critical for an ideal phylogenetic marker to investigate the potential existence of distinct TeHV3 genogroups and their associated pathology. Fifteen captive tortoises presumptively diagnosed to be infected with TeHVs or carrying compatible lesions on the basis of either the presence of intranuclear inclusions (presumptively infected) and/or diphtheronecrotic stomatitis-glossitis or pneumonia (compatible lesions) were selected for the study. Viral isolation, TeHV identification, phylogenetic analysis and pathological characterization of the associated lesions, were performed. Our results revealed 1) the existence of at least two distinct TeHV3 genogroups apparently associated with different pathologies in tortoises and 2) the first evidence for a putative homologous recombination event having occurred in a chelonian herpesvirus. This novel information is not only fundamental for the genetic characterization of this virus but is also critical to lay the groundwork for an improved understanding of host-pathogen interactions in chelonians and contribute to tortoise conservation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0134897PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4526542PMC
May 2016

The immune response of bovine mammary epithelial cells to live or heat-inactivated Mycoplasma bovis.

Vet Microbiol 2015 Sep 9;179(3-4):336-40. Epub 2015 Jul 9.

Veterinary Physiology, Vetsuisse Faculty University of Bern, Experimental station, Route de la Tioleyre 4, 1725 Posieux, Switzerland. Electronic address:

Mycoplasma bovis is an emerging bacterial agent causing bovine mastitis. Although these cell wall-free bacteria lack classical virulence factors, they are able to activate the immune system of the host. However, effects on the bovine mammary immune system are not yet well characterized and detailed knowledge would improve the prevention and therapy of mycoplasmal mastitis. The aim of this study was to investigate the immunogenic effects of M. bovis on the mammary gland in an established primary bovine mammary epithelial cell (bMEC) culture system. Primary bMEC of four different cows were challenged with live and heat-inactivated M. bovis strain JF4278 isolated from acute bovine mastitis, as well as with the type strain PG45. The immune response was evaluated 6 and 24h after mycoplasmal challenge by measuring the relative mRNA expression of selected immune factors by quantitative PCR. M. bovis triggered an immune response in bMEC, reflected by the upregulation of tumor necrosis factor-α, interleukin(IL)-1β, IL-6, IL-8, lactoferrin, Toll-like receptor-2, RANTES, and serum amyloid A mRNA. Interestingly, this cellular reaction was only observed in response to live, but not to heat-inactivated M. bovis, in contrast to other bacterial pathogens of mastitis such as Staphylococcus aureus. This study provides evidence that bMEC exhibit a strong inflammatory reaction in response to live M. bovis. The lack of a cellular response to heat-inactivated M. bovis supports the current hypothesis that mycoplasmas activate the immune system through secreted secondary metabolites.
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http://dx.doi.org/10.1016/j.vetmic.2015.07.007DOI Listing
September 2015

Invasion and persistence of Mycoplasma bovis in embryonic calf turbinate cells.

Vet Res 2015 May 15;46:53. Epub 2015 May 15.

Institute of Veterinary Bacteriology, Vetsuisse Faculty, University of Bern, Bern, Switzerland.

Mycoplasma bovis is a wall-less bacterium causing bovine mycoplasmosis, a disease showing a broad range of clinical manifestations in cattle. It leads to enormous economic losses to the beef and dairy industries. Antibiotic treatments are not efficacious and currently no efficient vaccine is available. Moreover, mechanisms of pathogenicity of this bacterium are not clear, as few virulence attributes are known. Microscopic observations of necropsy material suggest the possibility of an intracellular stage of M. bovis. We used a combination of a gentamicin protection assay, a variety of chemical treatments to block mycoplasmas entry in eukaryotic cells, and fluorescence and transmission electron microscopy to investigate the intracellular life of M. bovis in calf turbinate cells. Our findings indicate that M. bovis invades and persists in primary embryonic calf turbinate cells. Moreover, M. bovis can multiply within these cells. The intracellular phase of M. bovis may represent a protective niche for this pathogen and contribute to its escape from the host's immune defense as well as avoidance of antimicrobial agents.
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http://dx.doi.org/10.1186/s13567-015-0194-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4432498PMC
May 2015

Mycoplasma bovis infections in Swiss dairy cattle: a clinical investigation.

Acta Vet Scand 2015 Feb 22;57:10. Epub 2015 Feb 22.

Clinic for Ruminants, Vetsuisse Faculty, University of Berne, Bremgartenstrasse 109a, PO Box 8466, , CH-3001, Berne, Switzerland.

Mycoplasma bovis causes mastitis in dairy cows and is associated with pneumonia and polyarthritis in cattle. The present investigation included a retrospective case-control study to identify potential herd-level risk factors for M. bovis associated disease, and a prospective cohort study to evaluate the course of clinical disease in M. bovis infected dairy cattle herds in Switzerland. Eighteen herds with confirmed M. bovis cases were visited twice within an average interval of 75 d. One control herd with no history of clinical mycoplasmosis, matched for herd size, was randomly selected within a 10 km range for each case herd. Animal health data, production data, information on milking and feeding-management, housing and presence of potential stress- factors were collected. Composite quarter milk samples were aseptically collected from all lactating cows and 5% of all animals within each herd were sampled by nasal swabs. Organ samples of culled diseased cows were collected when logistically possible. All samples were analyzed by real-time polymerase chain reaction (PCR). In case herds, incidence risk of pneumonia, arthritis and clinical mastitis prior to the first visit and incidence rates of clinical mastitis and clinical pneumonia between the two visits was estimated. Logistic regression was used to identify potential herd-level risk factors for M. bovis infection. In case herds, incidence risk of M. bovis mastitis prior to the first visit ranged from 2 to 15%, whereas 2 to 35% of the cows suffered from clinical pneumonia within the 12 months prior to the first herd visit. The incidence rates of mycoplasmal mastitis and clinical pneumonia between the two herd visits were low in case herds (0-0.1 per animal year at risk and 0.1-0.6 per animal year at risk, respectively). In the retrospective-case-control study high mean milk production, appropriate stimulation until milk-let-down, fore-stripping, animal movements (cattle shows and trade), presence of stress-factors, and use of a specific brand of milking equipment, were identified as potential herd-level risk factors. The prospective cohort study revealed a decreased incidence of clinical disease within three months and prolonged colonization of the nasal cavity by M. bovis in young stock.
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http://dx.doi.org/10.1186/s13028-015-0099-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4347908PMC
February 2015

Cyto-adherence of Mycoplasma mycoides subsp. mycoides to bovine lung epithelial cells.

BMC Vet Res 2015 Feb 7;11:27. Epub 2015 Feb 7.

International Livestock Research Institute, P. O. Box 30709-00100, Nairobi, Kenya.

Background: Mycoplasma mycoides subsp. mycoides (Mmm) is the causative agent of contagious bovine pleuropneumonia (CBPP), a respiratory disease of cattle, whereas the closely related Mycoplasma mycoides subsp. capri (Mmc) is a goat pathogen. Cyto-adherence is a crucial step in host colonization by mycoplasmas and subsequent pathogenesis. The aim of this study was to investigate the interactions between Mmm and mammalian host cells by establishing a cyto-adherence flow cytometric assay and comparing tissue and species specificity of Mmm and Mmc strains.

Results: There were little significant differences in the adherence patterns of eight different Mmm strains to adult bovine lung epithelial cells. However, there was statistically significant variation in binding to different host cells types. Highest binding was observed with lung epithelial cells, intermediate binding with endothelial cells and very low binding with fibroblasts, suggesting the presence of effective adherence of Mmm on cells lining the airways of the lung, which is the target organ for this pathogen, possibly by high expression of a specific receptor. However, binding to bovine fetal lung epithelial cells was comparably low; suggesting that the lack of severe pulmonary disease seen in many infected young calves can be explained by reduced expression of a specific receptor.

Conclusions: Mmm bound with high efficiency to adult bovine lung cells and less efficiently to calves or goat lung cells. The data show that cyto-adherence of Mmm is species- and tissue- specific confirming its role in colonization of the target host and subsequent infection and development of CBPP.
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http://dx.doi.org/10.1186/s12917-015-0347-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4336739PMC
February 2015

Virulence, persistence and dissemination of Mycoplasma bovis.

Vet Microbiol 2015 Aug 2;179(1-2):15-22. Epub 2015 Mar 2.

Institute of Veterinary Bacteriology, Vetsuisse Faculty, University of Bern, Bern, Switzerland. Electronic address:

Bovine mycoplasmosis due to Mycoplasma bovis causes several important bovine diseases such as pneumonia, mastitis, arthritis, otitis, genital disorders or keratoconjunctivitis. Variable surface lipoproteins, adhesion, invasion of host cells, modulation of the host immune system, biofilm formation and the release of secondary metabolites like hydrogen peroxide, as well as synergistic infections with other bacterial or viral pathogens are among the more significantly studied characteristics of the bacterium. The aim of this review is to summarize the current knowledge regarding the virulence of M. bovis and additionally, factors contributing to the dissemination and persistence of this pathogen in the bovine host will be discussed.
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http://dx.doi.org/10.1016/j.vetmic.2015.02.024DOI Listing
August 2015

Tularemia among free-ranging mice without infection of exposed humans, Switzerland, 2012.

Emerg Infect Dis 2015 Jan;21(1):133-5

The animals primarily infected by Francisella tularensis are rapidly consumed by scavengers, hindering ecologic investigation of the bacterium. We describe a 2012 natural tularemia epizootic among house mice in Switzerland and the assessment of infection of exposed humans. The humans were not infected, but the epizootic coincided with increased reports of human cases in the area.
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http://dx.doi.org/10.3201/eid2101.140906DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4285241PMC
January 2015

First report of a multidrug-resistant Klebsiella pneumoniae of sequence type 11 causing sepsis in a free-ranging beaver (Castor fiber).

Environ Microbiol Rep 2015 Apr 12;7(2):351-3. Epub 2015 Feb 12.

Institute of Veterinary Bacteriology, Vetsuisse Faculty, University of Bern, Bern, Switzerland.

Klebsiella pneumoniae of sequence type (ST) 11 is a hyper-epidemic nosocomial clone spreading worldwide among humans and also emerging in pets. In this report, we describe a clinical case of fatal sepsis due to this multidrug-resistant (MDR) pathogen in a Eurasian beaver. The isolate showed resistance to six different classes of antimicrobials including third generation cephalosporins and fluoroquinolones. This is the first report describing the detection of a MDR K. pneumoniae ST11 in a free-ranging animal. Our finding highlights the potential for environmental dissemination of hyper-epidemic clones of K. pneumoniae and possible spread in wildlife and cause epizootics.
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http://dx.doi.org/10.1111/1758-2229.12257DOI Listing
April 2015

Identification of amino acid substitutions with compensational effects in the attachment protein of canine distemper virus.

J Virol 2014 Jul 7;88(14):8057-64. Epub 2014 May 7.

Centre for Fish and Wildlife Health (FIWI), Vetsuisse Faculty, University of Bern, Bern, Switzerland Institute of Veterinary Bacteriology (IVB), Vetsuisse Faculty, University of Bern, Bern, Switzerland

The hemagglutinin (H) gene of canine distemper virus (CDV) encodes the receptor-binding protein. This protein, together with the fusion (F) protein, is pivotal for infectivity since it contributes to the fusion of the viral envelope with the host cell membrane. Of the two receptors currently known for CDV (nectin-4 and the signaling lymphocyte activation molecule [SLAM]), SLAM is considered the most relevant for host susceptibility. To investigate how evolution might have impacted the host-CDV interaction, we examined the functional properties of a series of missense single nucleotide polymorphisms (SNPs) naturally accumulating within the H-gene sequences during the transition between two distinct but related strains. The two strains, a wild-type strain and a consensus strain, were part of a single continental outbreak in European wildlife and occurred in distinct geographical areas 2 years apart. The deduced amino acid sequence of the two H genes differed at 5 residues. A panel of mutants carrying all the combinations of the SNPs was obtained by site-directed mutagenesis. The selected mutant, wild type, and consensus H proteins were functionally evaluated according to their surface expression, SLAM binding, fusion protein interaction, and cell fusion efficiencies. The results highlight that the most detrimental functional effects are associated with specific sets of SNPs. Strikingly, an efficient compensational system driven by additional SNPs appears to come into play, virtually neutralizing the negative functional effects. This system seems to contribute to the maintenance of the tightly regulated function of the H-gene-encoded attachment protein. Importance: To investigate how evolution might have impacted the host-canine distemper virus (CDV) interaction, we examined the functional properties of naturally occurring single nucleotide polymorphisms (SNPs) in the hemagglutinin gene of two related but distinct strains of CDV. The hemagglutinin gene encodes the attachment protein, which is pivotal for infection. Our results show that few SNPs have a relevant detrimental impact and they generally appear in specific combinations (molecular signatures). These drastic negative changes are neutralized by compensatory mutations, which contribute to maintenance of an overall constant bioactivity of the attachment protein. This compensational mechanism might reflect the reaction of the CDV machinery to the changes occurring in the virus following antigenic variations critical for virulence.
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http://dx.doi.org/10.1128/JVI.00454-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4097785PMC
July 2014

A genomic perspective on a new bacterial genus and species from the Alcaligenaceae family, Basilea psittacipulmonis.

BMC Genomics 2014 Mar 1;15:169. Epub 2014 Mar 1.

Genomic Research Laboratory, Department of Internal Medicine, Service of Infectious Diseases, Geneva University Hospitals, Gabrielle-Perret-Gentil 4, CH-1211 Geneva 14, Switzerland.

Background: A novel Gram-negative, non-haemolytic, non-motile, rod-shaped bacterium was discovered in the lungs of a dead parakeet (Melopsittacus undulatus) that was kept in captivity in a petshop in Basel, Switzerland. The organism is described with a chemotaxonomic profile and the nearly complete genome sequence obtained through the assembly of short sequence reads.

Results: Genome sequence analysis and characterization of respiratory quinones, fatty acids, polar lipids, and biochemical phenotype is presented here. Comparison of gene sequences revealed that the most similar species is Pelistega europaea, with BLAST identities of only 93% to the 16S rDNA gene, 76% identity to the rpoB gene, and a similar GC content (~43%) as the organism isolated from the parakeet, DSM 24701 (40%). The closest full genome sequences are those of Bordetella spp. and Taylorella spp. High-throughput sequencing reads from the Illumina-Solexa platform were assembled with the Edena de novo assembler to form 195 contigs comprising the ~2 Mb genome. Genome annotation with RAST, construction of phylogenetic trees with the 16S rDNA (rrs) gene sequence and the rpoB gene, and phylogenetic placement using other highly conserved marker genes with ML Tree all suggest that the bacterial species belongs to the Alcaligenaceae family. Analysis of samples from cages with healthy parakeets suggested that the newly discovered bacterial species is not widespread in parakeet living quarters.

Conclusions: Classification of this organism in the current taxonomy system requires the formation of a new genus and species. We designate the new genus Basilea and the new species psittacipulmonis. The type strain of Basilea psittacipulmonis is DSM 24701 (= CIP 110308 T, 16S rDNA gene sequence Genbank accession number JX412111 and GI 406042063).
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http://dx.doi.org/10.1186/1471-2164-15-169DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4028982PMC
March 2014

Epidemiology of brucellosis and q Fever in linked human and animal populations in northern togo.

PLoS One 2013 12;8(8):e71501. Epub 2013 Aug 12.

Department of Epidemiology and Public Health, Swiss Tropical and Public Health Institute, Basel, Switzerland ; University of Basel, Basel, Switzerland.

Background: Although brucellosis (Brucella spp.) and Q Fever (Coxiella burnetii) are zoonoses of global importance, very little high quality data are available from West Africa.

Methods/principal Findings: A serosurvey was conducted in Togo's main livestock-raising zone in 2011 in 25 randomly selected villages, including 683 people, 596 cattle, 465 sheep and 221 goats. Additionally, 464 transhumant cattle from Burkina Faso were sampled in 2012. The serological analyses performed were the Rose Bengal Test and ELISA for brucellosis and ELISA and the immunofluorescence assay (IFA) for Q Fever Brucellosis did not appear to pose a major human health problem in the study zone, with only 7 seropositive participants. B. abortus was isolated from 3 bovine hygroma samples, and is likely to be the predominant circulating strain. This may explain the observed seropositivity amongst village cattle (9.2%, 95%CI:4.3-18.6%) and transhumant cattle (7.3%, 95%CI:3.5-14.7%), with an absence of seropositive small ruminants. Exposure of livestock and people to C. burnetii was common, potentially influenced by cultural factors. People of Fulani ethnicity had greater livestock contact and a significantly higher seroprevalence than other ethnic groups (Fulani: 45.5%, 95%CI:37.7-53.6%; non-Fulani: 27.1%, 95%CI:20.6-34.7%). Appropriate diagnostic test cut-off values in endemic settings requires further investigation. Both brucellosis and Q Fever appeared to impact on livestock production. Seropositive cows were more likely to have aborted a foetus during the previous year than seronegative cows, when adjusted for age. This odds was 3.8 times higher (95%CI: 1.2-12.1) for brucellosis and 6.7 times higher (95%CI: 1.3-34.8) for Q Fever.

Conclusions: This is the first epidemiological study of zoonoses in Togo in linked human and animal populations, providing much needed data for West Africa. Exposure to Brucella and C. burnetii is common but further research is needed into the clinical and economic impact.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0071501PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3741174PMC
April 2014

Francisella tularensis infection in a stone marten (Martes foina) without classic pathological lesions consistent with tularemia.

J Vet Diagn Invest 2013 Jul 14;25(4):519-21. Epub 2013 May 14.

Institute for Veterinary Bacteriology, Vetsuisse Faculty, Bern, Switzerland.

The current report describes the isolation and typing of a strain of Francisella tularensis, the causative agent of tularemia, from the spleen of a stone marten (Martes foina) showing no classic lesions consistent with the disease. The identification of this bacterium, belonging to the World Health Organization risk 3 category and considered to have a low infectious dose, could be performed only because of an ongoing project screening F. tularensis in the environment sensu lato. The findings described herein should alert diagnostic laboratories of the possible presence of F. tularensis in clinical samples in countries where tularemia is endemic even in cases with no consistent anamnesis and from unsuspected animal species.
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http://dx.doi.org/10.1177/1040638713489124DOI Listing
July 2013

Molecular epidemiology and antibiotic susceptibility of livestock Brucella melitensis isolates from Naryn Oblast, Kyrgyzstan.

PLoS Negl Trop Dis 2013 28;7(2):e2047. Epub 2013 Feb 28.

State Veterinary Department, Bishkek, Kyrgyzstan.

The incidence of human brucellosis in Kyrgyzstan has been increasing in the last years and was identified as a priority disease needing most urgent control measures in the livestock population. The latest species identification of Brucella isolates in Kyrgyzstan was carried out in the 1960s and investigated the circulation of Brucella abortus, B. melitensis, B. ovis, and B. suis. However, supporting data and documentation of that experience are lacking. Therefore, typing of Brucella spp. and identification of the most important host species are necessary for the understanding of the main transmission routes and to adopt an effective brucellosis control policy in Kyrgyzstan. Overall, 17 B. melitensis strains from aborted fetuses of sheep and cattle isolated in the province of Naryn were studied. All strains were susceptible to trimethoprim-sulfamethoxazole, gentamicin, rifampin, ofloxacin, streptomycin, doxycycline, and ciprofloxacin. Multilocus variable number tandem repeat analysis showed low genetic diversity. Kyrgyz strains seem to be genetically associated with the Eastern Mediterranean group of the Brucella global phylogeny. We identified and confirmed transmission of B. melitensis to cattle and a close genetic relationship between B. melitensis strains isolated from sheep sharing the same pasture.
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http://dx.doi.org/10.1371/journal.pntd.0002047DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3584998PMC
July 2013

Herd-specific strains of Mycoplasma bovis in outbreaks of mycoplasmal mastitis and pneumonia.

Vet Microbiol 2012 Jun 15;157(3-4):363-8. Epub 2012 Jan 15.

Institute of Veterinary Bacteriology, Vetsuisse Faculty, University of Bern, Bern, Switzerland.

Mycoplasma bovis causes severe economic losses in livestock production, particularly on the Northern American continent and more recently also in continental Europe. The aim of the current study was to evaluate whether the recently emerging outbreaks were due to a particular clone or strain of M. bovis or whether these outbreaks are due to multiple infectious strains of M. bovis. The study is based on the analysis M. bovis isolated from cattle of herds with outbreaks of mycoplasmal mastitis or pneumonia from geographically non related parts of Switzerland. M. bovis isolates were typed by insertion sequence (IS) element analysis based upon ISMbov1 and ISMbov2 southern-blot hybridization. We observed a strong divergence of M. bovis strains among affected herds which mostly were herd specific. This argues against the assumption that a recent infiltration of a particular clone of M. bovis is the cause of the perilous emerging outbreaks. The study suggests that transmission occurs from animal to animal most probably via milk.
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http://dx.doi.org/10.1016/j.vetmic.2012.01.006DOI Listing
June 2012