Isolation of non-tuberculous mycobacteria from pastoral ecosystems of Uganda: Public Health significance

<p>Abstract</p> <p>Background</p> <p>The importance of non-tuberculous mycobacteria (NTM) infections in humans and animals in sub-Saharan Africa at the human-environment-livestock-wildlife interface has recently received increased attention. NTM are environmental opportunistic pathogens of humans and animals. Recent studies in pastoral ecosystems of Uganda detected NTM in humans with cervical lymphadenitis and cattle with lesions compatible with bovine tuberculosis. However, little is known about the source of these mycobacteria in Uganda. The aim of this study was to isolate and identify NTM in the environment of pastoral communities in Uganda, as well as assess the potential risk factors and the public health significance of NTM in these ecosystems.</p> <p>Method</p> <p>A total of 310 samples (soil, water and faecal from cattle and pigs) were examined for mycobacteria. Isolates were identified by the INNO-Lipa test and by 16S rDNA sequencing. Additionally, a questionnaire survey involving 231 pastoralists was conducted during sample collection. Data were analysed using descriptive statistics followed by a multivariable logistic regression analysis.</p> <p>Results</p> <p>Forty-eight isolates of NTM were detected; 25.3% of soil samples, 11.8% of water and 9.1% from animal faecal samples contained mycobacteria. Soils around water sources were the most contaminated with NTM (29.8%). Of these samples, <it>M. fortuitum-peregrinum </it>complex, <it>M. avium </it>complex, <it>M. gordonae</it>, and <it>M. nonchromogenicum </it>were the most frequently detected mycobacteria. Drinking untreated compared to treated water (OR = 33), use of valley dam versus stream water for drinking and other domestic use (OR = 20), sharing of water sources with wild primates compared to antelopes (OR = 4.6), sharing of water sources with domestic animals (OR = 5.3), and close contact with cattle or other domestic animals (OR = 13.8) were the most plausible risk factors for humans to come in contact with NTM in the environment.</p> <p>Conclusions</p> <p>The study detected a wide range of potentially pathogenic NTM from the environment around the pastoral communities in Uganda. Drinking untreated water and living in close contact with cattle or other domestic animals may be risk factors associated with the possibility of humans and animals acquiring NTM infections from these ecosystems.</p

Early Responses of Natural Killer Cells in Pigs Experimentally Infected with 2009 Pandemic H1N1 Influenza A Virus

Natural killer (NK) cells are important players in the innate immune response against influenza A virus and the activating receptor NKp46, which binds hemagglutinin on the surface of infected cells, has been assigned a role in this context. As pigs are natural hosts for influenza A viruses and pigs possess both NKp46− and NKp46+ NK cells, they represent a good animal model for studying the role of the NKp46 receptor during influenza. We explored the role of NK cells in piglets experimentally infected with 2009 pandemic H1N1 influenza virus by flow cytometric analyses of cells isolated from blood and lung tissue and by immunostaining of lung tissue sections. The number of NKp46+ NK cells was reduced while NKp46− NK cells remained unaltered in the blood 1–3 days after infection. In the lungs, the intensity of NKp46 expression on NK cells was increased during the first 3 days, and areas where influenza virus nucleoprotein was detected were associated with increased numbers of NKp46+ NK cells when compared to uninfected areas. NKp46+ NK cells in the lung were neither found to be infected with influenza virus nor to be undergoing apoptosis. The binding of porcine NKp46 to influenza virus infected cells was verified in an in vitro assay. These data support the involvement of porcine NKp46+ NK cells in the local immune response against influenza viru

Human to animal transmission of influenza A(H1N1)pdm09 in a turkey breeder flock in Norway

Introduction: Routine surveillance samples disclosed seropositivity to influenza A virus (IAV) in a Norwegian turkey breeder flock. Simultaneous reports of influenza-like symptoms in farm workers and a laboratory confirmed influenza A(H1N1)pdm09 (H1N1pdm09) infection in one person led to the suspicion of a H1N1pdm09 infection in the turkeys. Animals and methods: H1N1pdm09 infection was confirmed by a positive haemaggutinin inhibition test using H1N1pdm09 antigens, and detection of H1N1pdm09 nucleic acid in reproductive organs of turkey hens. The flock showed no clinical signs except for a temporary drop in egg production. Previous reports of H1N1pdm09 infection in turkeys suggested human-to-turkey transmission (anthroponosis) during artificial insemination. Results and discussion: The flock remained seropositive to IAV and the homologous H1N1pdm09 antigen throughout the following 106 days, with decreasing seroprevalence over time. IAV was not detected in fertilised eggs or in turkey poults from the farm, however, maternally derived antibodies against H1N1pdm09 were found in egg yolks and in day-old poults. Genetic analyses of haemagglutinin gene sequences from one of the infected farm workers and turkeys revealed a close phylogenetic relationship, and confirmed human-to-turkey virus transmission

Early Responses of Natural Killer Cells in Pigs Experimentally Infected with 2009 Pandemic H1N1 Influenza A Virus

<div><p>Natural killer (NK) cells are important players in the innate immune response against influenza A virus and the activating receptor NKp46, which binds hemagglutinin on the surface of infected cells, has been assigned a role in this context. As pigs are natural hosts for influenza A viruses and pigs possess both NKp46⁻ and NKp46⁺ NK cells, they represent a good animal model for studying the role of the NKp46 receptor during influenza. We explored the role of NK cells in piglets experimentally infected with 2009 pandemic H1N1 influenza virus by flow cytometric analyses of cells isolated from blood and lung tissue and by immunostaining of lung tissue sections. The number of NKp46⁺ NK cells was reduced while NKp46⁻ NK cells remained unaltered in the blood 1–3 days after infection. In the lungs, the intensity of NKp46 expression on NK cells was increased during the first 3 days, and areas where influenza virus nucleoprotein was detected were associated with increased numbers of NKp46⁺ NK cells when compared to uninfected areas. NKp46⁺ NK cells in the lung were neither found to be infected with influenza virus nor to be undergoing apoptosis. The binding of porcine NKp46 to influenza virus infected cells was verified in an <i>in vitro</i> assay. These data support the involvement of porcine NKp46⁺ NK cells in the local immune response against influenza virus.</p></div

Staining for apoptosis in the lungs.

<p>Lung tissue sections from animals infected with influenza A virus (<i>n</i> = 12) were stained with immunofluorescence markers against (<b>A</b>) NKp46(green), (<b>B</b>) influenza A NP(red) and (<b>C</b>) the apoptosis marker caspase-3(blue). (<b>D</b>) Overlay displaying simultaneously influenza A virus NP⁺ and caspase-3⁺ cells as purple (arrows). Representative of virus infected bronchiole at day 1 pi. Immunofluorescence staining, 400x.</p

NKp46⁺ cells in the lungs of influenza virus infected pigs.

<p>Lung tissue sections from pigs infected with influenza A virus and control pigs were stained with immunofluorescence markers for cytokeratin (blue), NKp46 (green) and influenza A virus NP (red). NKp46⁺ cells were counted in areas were influenza A virus NP was (<b>A</b>) detected and (<b>B</b>) not detected. Representative pictures taken from the same animal on day 1 pi are shown. Arrows point at NKp46⁺ cells. Immunofluorescence staining, 200x. (<b>C</b>) Plot shows number of NKp46⁺ cells per 0,1 mm² in sections (<i>n</i> = 24 per animal) from control animals (<i>n</i> = 6) and in areas with and without virus in infected animals (<i>n</i> = 4 per day) calculated as described in <i>Material and Methods</i>. Groups with different letters differ significantly (<i>p</i>≤0.05). (<b>D</b>) NKp46⁺ cells in the lumen of a bronchus (BL). Arrows point at the epithelial lining. Representative picture of luminal exudate, taken from an infected animal on day 2 pi. Insert shows NKp46⁺ and influenza A virus NP⁺ cell in the lung tissue of an infected animal on day 1 pi. Immunofluorescence staining, 400x.</p

Detection of IFN-γ and TNF in lung tissue.

<p>(<b>A</b>) Intracellular IFN-γ was analysed in lung mononuclear cells from influenza A virus infected animals and control animals by flow cytometry. IFN-γ ⁺ cells were gated among live lymphocytes. Plots show representative isotype control (left) and IFN-γ staining (right) from the same infected animal on day 3 pi (<b>B</b>) Percentages of IFN-γ⁺ cells obtained by flow cytometry in control animals (<i>n</i> = 8) and infected animals (<i>n</i> = 4 per day). Data from one animal at day 1 is missing due to too few cells isolated. (<b>C</b>) Gene expression for IFN-γ and TNF mRNA in infected animals (<i>n</i> = 4 per day) was calculated as relative values to the household gene GADPH and to mRNA levels of the target gene in a control group (<i>n</i> = 4). Each symbol represents one infected animal, different symbols represents animals sacrificed the same day. Values above 1 indicate an up regulation, whereas values below 1 indicate a down regulation of the target gene.</p