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Bioaerosol Measurement in Animal Environments


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Measuring bioaerosols in animal facilities is challenging. This presentation includes a classroom lecture and also a hands-on workshop or lab portion.

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Bioaerosol Measurement in Animal Environments

  1. 1. Bioaerosol Measurement in Animal Environments Continuing Professional Development Lingjuan Wang-Li & Otto D. Simmons IIIDepartment of Biological & Agricultural Engineering North Carolina State University 1
  2. 2. Bioaerosol Measurement in Animal Environments Part I: Classroom Lecture Part II: Bioaerosol Sampling: Demonstration & Hands-on Practice 2
  3. 3. Part I: Classroom Lecture 3
  4. 4. Overview: • Bioaerosol fundamentals • Bioaerosol in animal environments • Bioaerosol sampling • Bioaerosol sampler selection • Biological analyses 4
  5. 5. Bioaerosol Fundamentals 5
  6. 6. Bioaerosol Fundamentals: Definitions• Aerosol: a suspension of solid/liquid particles in a gas  Includes both the particles and the suspending gas, e.g. air• Bioaerosol: an aerosol of biological origin, or  Particles of biological origin suspended in the air• Particulate matter (PM): the generic term for a broad class of chemically and physically diverse substances that exist as discrete particle in liquid droplets or solids forms in the air (EPA’s definition)  PM2.5/PM10 : criteria pollutant - NAAQS 6
  7. 7. Bioaerosol Fundamentals: Airborne Microbes & Aerosols • Airborne transmission is possible for essentially all classes of microbes: viruses, bacteria, fungi, and protozoans • Any respiratory pathogen able to survive aerosolization and air transport is considered a potential cause of airborne disease 7
  8. 8. Bioaerosol Fundamentals: Bioaerosol Classification • Viruses, parasites • Living organisms  bacteria  fungi • Parts of products of organisms  fungal spores  pollen  endotoxin  allergens from dogs, cats and insects 8
  9. 9. Bioaerosol Fundamentals:Particle size and natural background concentration of bioaerosols:Source: Hinds, W.C. Aerosol Technology: Properties, Behavior, and Measurement of Airborne Particles, 2 nd edition.• Bioaerosol particles often occur as agglomerates, as clusters of organisms in droplets or attached to other airborne debris• Bioaerosols can be subdivided into two groups:  viable: living organisms  nonviable: dead organism, pollen, animal dander, etc. 9
  10. 10. Bioaerosol Fundamentals: Bacteria Bacteria are single-celled organisms with size from 0.3 ~ 10 µ m • spherical or rod shaped • occur as clusters or chains • pathogens – cause human disease •ambient – colonize water or soil and released as aerosols when the water or soil is disturbed •indoor – colonize accumulations of moisture in ventilation systems and become aerosolized by air currentsSource for the photo: or vibration 10
  11. 11. Bioaerosol Fundamentals:Bacteria: two groups based upon the ability of the cell wall to retain crystal violet dye• Gram-positive: retain the dye;  lack the outer membrane  Most pathogenic bacteria – Gram-positive• Gram-negative: cannot retain the dye  Escherichia coli, SalmonellaEndotoxins: a structural component in bacteria released when bacteria are lysed• Chemically stable and heat resistant 11
  12. 12. Bioaerosol Fundamentals: Fungi Fungi: a unique group of organism – 70,000 have been identifiedYeast cell: single celled organisms Mold - hyphae Fungal hyphae • Most fungi disperse by releasing spores into the air • Fungal spores often occur as individual particles Source for the photos:Fungal spores: 0.5 – 30 µ m 12
  13. 13. Bioaerosol Fundamentals: Viruses Viruses are intracellular parasites that can reproduce only inside a host cell a cluster of influenza viruses viruses that cause tobacco mosaic disease in tobacco plants • naked viruses: from 0.02 – 0.3 µ m • airborne viruses – part of droplet nuclei or attached to other particles • transmitted by direct contact, or by inhalation of aerosolized viruses • aerosolization by coughing, sneezing or talking • can survive for weeks on fabric or carpets Source for the photos: 13
  14. 14. Bioaerosol Fundamentals: Pollen Pollen grains: 10 – 100 µ m with most between 25 - 50 µ m • near spherical particles • transmission of genetic material • anemophilous (wind-pollinated) plants – produce abundant bioaerosol pollen – wind- borne pollen • insect-pollinated plants – produce sticky pollen that is not readily aerosolizedPollen from a variety of common plants: • causes allergic diseases of the uppersunflower, morning glory, hollyhock, etc. airways (hay fever) Photo source: 14
  15. 15. Bioaerosol Fundamentals: Microbial Viability & Infectivity • Viability (survival): ability to replicate • Infectivity: ability to cause infection 15
  16. 16. Bioaerosol Fundamentals: Aerosol Factors Influencing Airborne Infection• Particle size: <5 um "droplet nuclei" from coughing & sneezing  Deposition site depends on particle size and hygroscopicity  Chemical composition of the aerosol particle• Relative humidity (RH): dessication (loss of moisture)• Temperature: generally greater inactivation at higher temp.• Sunlight: UV inactivation of microbes• Factors influencing air movement: winds, currents, mechanical air handlers, etc.• Seasonal factors: precipitation, air currents, pollen sources, etc.• Air pollution:  chemicals inactivating airborne microbes (OAF= Open Air Factor)  enhancing their ability to cause infection in a host 16
  17. 17. Bioaerosol Fundamentals: Some Common Airborne Infectious Diseases Virus Disease Bacteria Disease Adenovirus Respiratory Bordetella pertussis Whooping infection cough Herpesvirus Chicken pox Yersinia pestis Pneumonic plague Poxvirus Small pox Staphylococcus aureus Wound infection Togavirus Rubella Mycobacterium TB tuberculosis Orthomyxovirus Influenza Legionella pneumophila Legionellosis Paramyxovirus Measles, mumps Bacillus anthracis Anthrax Rhabdovirus Newcastle Coxiella burnetii Q fever 17
  18. 18. Bioaerosol Fundamentals: Diseases Caused by Bioaerosols: Hypersensitivity or Allergic Diseases Result from exposure to antigens (of indoor bioaerosols) that stimulate an allergic response by the bodys immune system. • Susceptiblity varies among people. • Diseases usually are diagnosed by a physician. • Once an individual has developed a hypersensitivity disease, a very small amount of the antigen may cause a severe reaction. • Hypersensitivity diseases account for most of the health problems due to indoor bioaerosols 18
  19. 19. Bioaerosol Fundamentals: 19
  20. 20. Bioaerosol Fundamentals: Regions of the Respiratory System The cellular composition and anatomy of the respiratory system influence particle deposition • Nasopharynx Region: the head region, including the nose, mouth, pharynx, and larynx • Tracheobronchial Region: includes the trachea, bronchi, and bronchioles • Pulmonary (Alveolar) Region: comprised of the alveoli; the exchange of oxygen and carbon dioxide through the process of respiration occurs in the alveolar region 20
  21. 21. Bioaerosol Fundamentals:Regional Deposition in Respiratory Tract vs. Particle Size 21
  22. 22. Bioaerosol Fundamentals: Aerosols & Respiratory Deposition Aerosols > 5 microns in diameter are removed in the upper respiratory tract, especially the nose. • Particles are brought to the pharynx by mucociliary activity of the upper respiratory epithelial mucosa, where they are expectorated or swallowed. Swallowed particles containing enteric microbes can initiate enteric infections 22
  23. 23. Bioaerosol Fundamentals: Aerosols & Respiratory Deposition Particles <5 microns in diameter, esp. 1‑ 3 microns diameter, penetrate to the lower respiratory tract • Can be deposited in the bronchioles, alveolar ducts and alveoli • Deposition efficiency in lower respiratory tract is ~50% for particles 1‑ 2 microns diameter. • Can also be deposited in the lower respiratory tract, especially particles <0.25 microns dia. • Particles deposited in the lower respiratory tract can be phagocytized by respiratory (alveolar) macrophages can be destroyed or carried to the ciliary escalator, where they are transported upward to the pharynx 23
  24. 24. Bioaerosol Fundamentals: Hygroscopicity & Aerosol Deposition in the Respiratory Tract When inhaled, aerosol particles derived from aqueous fluids pick up moisture (water) while traveling in the respiratory passageways, thereby increasing in size.  Increased size changes deposition site H2O H2O H2O 24
  25. 25. Bioaerosol in Animal Environments (Important issue, and yet understudied) 25
  26. 26. Bioaerosol in Animal Environments: Common Pathogenic Bioaerosols in Poultry & Pig housesSource: Cox, C.S. and C.M. Wathes. 1995. Bioaerosols Handbook. 26
  27. 27. Bioaerosol in Animal Environments: Common infectious disease of farm animals and pathogens Host diseases Factors implicated in causation pathogens environment pigs Atrophic rhinitis Bordetella bronchiseptica Crowding Pasteurella multocida Poor ventilation Enzootic Mycoplasma suipneumoniae Poor drainage, high pneumonia relative humidity cattle Diarrhea Rotavirus, E. Coli, etc. Weaning, hygiene, cold pneumonia Mycoplasma bovis, dispar Crowding, poor feeding Shipping fever P. haemolytica etc. High relative humidity, stress Environmental E. Coli, strep. uberis Contaminated bedding, mastitis stage of lactation horses Obstructive Mycropolyspora faeni Dusty feed and bedding, pulmonary disease poor ventilation Aspergillus fumigatusSource: Cox, C.S. and C.M. Wathes. 1995. Bioaerosols Handbook. 27
  28. 28. Bioaerosol Sampling 28
  29. 29. General Considerations:• Why? – sampling objectives• What? – measurement variables• Where? – take representative samples: (sampling locations, number of sites)• When? – frequency of sampling (statistical replicates)• How? – sampler selection & sampling processes/steps• Results? – determination of concentration and/or emission rate 29
  30. 30. Sampling Objectives: • Verify and quantify the presence of bioaerosols (specific species or total bioaerosol?) • Identify their sources for control • Evaluate the effectiveness of control measures • Others (fate and transport …) 30
  31. 31. Taking Representative Samples: Temporal and spatial variations in bioaerosol speciation and concentrations: • Sampling locations: horizontal (building layout, ventilation system…) & vertical (animal or human height) • Number of sampling sites: statistical replicates • Frequency of sampling: diurnal, seasonal variations • Optima sampling duration: concentration dependent 31
  32. 32. Sampling Procedures: Step 1: Agar or nutrient broth preparation Step 2: Sampler flowrate calibration Step 3: Sample collection with a viable sampler Step 4: Sample transportation Step 5: Sample condition Step 6: Sample analysis 32
  33. 33. Result Analysis: Sample Concentration Determination N C= [cfu/m3 = colony-forming units /m3] Q*t • C = bioaerosol concentration in cfu/m3 • N = total bioaerosol counts on the agar plate (in the sample, #) • Q = sampling flowrate of the viable sampler (m3/min) • t = sampling duration (min.) 33
  34. 34. Result Analysis: Emission Rate Determination ER = concentration * ventilation rate • ER = emission rate of bioaerosol from animal housing in cfu/min • Concentration = in-house bioaerosol concentration in cfu/m3 • Ventilation rate = air flowrate of animal housing ventilation fans in m3/min 34
  35. 35. Bioaerosol Sampler Selection 35
  36. 36. Bioaerosol Samplers: Viable Sampling SystemsSize selective system Non-size- selective system Sampled air Sampled air Size selective sampling head, nozzles collecting medium (filter, or, agar collecting medium (filter, or, agar plate, or nutrient broth ) plate, or nutrient broth ) Calibrated flow Calibrated flow monitoring/control unit) monitoring/control unit) Air Air pump discharged pump discharged 36
  37. 37. Bioaerosol Samplers: Total Sampling Efficiency The overall sampling efficiency of a bioaerosol sampler:  the inlet sampling efficiency – the same as for non-bioaerosol sampling – depends on the size, shape and aerodynamics of the particles being sampled – first stage collection/deposition efficiency onto glass slides, a semisolid culture medium – second stage the biological aspect of sampling efficiency – depends on the sampling and removal of biological particles without altering their viability or biological activity – biological analysis to identify & quantify the biological particle presents – third stage None of the presently available samplers for culturable bioaerosols can be considered as reference method • Glass liquid impingers (AGI, HAM, MIL) • Six-stage Andersen impactor (AND) 37
  38. 38. Bioaerosol Samplers: Principles of Bioaerosol Collection Inertial impaction: the inertial of the particle forces its impaction onto a solid or semisolid impaction surface – a cultural medium, or an adhesive surface – be examined microscopically Single-stage impactors: the surface air sampler, PBI, SPI Cascade impactors: two or more impaction stages (the Anderson cascade impactor) Slit samplers: the impaction stage consists of one or more slits instead of one or more circle holes (CAS, NBS, BAS cultural plate samplers) 38
  39. 39. Bioaerosol Samplers: Principle of Collection - Inertial ImpactionImpaction ~ a special case of curvilinear motion ~ application in the collection and measurement of aerosol particlesAssumption: particles stick to the surfaceof the impaction plate once they hit it 39
  40. 40. Bioaerosol Samplers: Principle of Collection - Inertial ImpactionAssumptions: the flow velocity is uniform in the jet; the streamlines are arcs of a circle with centers at A Y τU 2 X Vr = τa r = r τU 2  2πr  π ∆ = Vr t =   = τU r  4U  2 ∆ πτU π EI = = = Stk h 2h 2 40
  41. 41. Bioaerosol Samplers: Principle of Collection - Inertial Impaction Stk. to characterize inertial impaction: The characterization dimension : 2 • the radius of the nozzle jet = Dj/2 for a τV ρp d p VC c circular jet Stk = = Dj 2 9ηD j • the jet half-width = W/2 for a rectangular jet 1.00 Collection efficiency 0.80 0.60 9ηD jStk 50 0.40 d 50 C c = 0.20 ρp V 0.00 0 2 4 6 8 10 Aerodynamic equivalent diameter 41
  42. 42. Aerosol Samplers In General: Sampler Fractional Efficiency Curve (FEC) Ideally, it is desired that all particles greater than a certain size are collected and all particles smaller than that size pass through – Cut-off size FEC: relates collection efficiency to the particle diameters Cut-point , cut-off size (d50): is the AED of the particle with 50% efficiency Slope: it the sharpness of the cut Aerodynamic equivalent diameter (µm) 42
  43. 43. Bioaerosol Samplers: Principle of Collection – Multi-stage Impaction Cut-points for different stages 43
  44. 44. Bioaerosol Samplers: Principles of Bioaerosol Collection Centrifugal inertial impaction: particle separation by centrifugal force in a radial geometry - the Reuter centrifugal sampler (BIO) Liquid impingement: the particles are collected by inertial impaction into a liquid, and particle diffusion within the bubbles (the AGI-4 and AGI-30 impingers) Tangential impinger: collects particles by inertial impaction and centrifugation (BioSampler SKC) 44
  45. 45. Bioaerosol Samplers: Principles of Bioaerosol CollectionFiltration: impaction, interception, diffusion, gravitationalsettling, etc. − particle physical properties, filter pore size,air flow Challenges: inlet – isokinetic sampling ?filter: dehydration effect – desiccation stress?Gravitational Settling: the least effective methods of bioaerosol collection – particle size, shape and airflow dictate thedeposition of particlesElectrostatic Precipitation: overcome some of physical damage caused by impinger, or impactor 45
  46. 46. Bioaerosol Samplers: Viable Samplers – Inertial ImpactorsAnderson single stage Anderson two-stage Anderson six-stage Stage with Petri- viable impactor viable impactor viable impactor dish 46
  47. 47. Bioaerosol Samplers: Viable Samplers – Impingers All glass AGI-30 liquid impinger Multistage all glass liquid impinger 47
  48. 48. Bioaerosol Samplers: Viable Samplers – Centrifugal Sampler “Aerojet” cyclone sampler RCS Biotest centrifugal sampler 48
  49. 49. Bioaerosol Samplers: High impact velocity can result in metabolic and structural injuries of the collected microorganisms 1 – 265 m/s Selection of sampler: cutoff size and the aerodynamic particle size 49
  50. 50. Bioaerosol Samplers: Selection of Sampler SAS samplers – portable one stage multiple-hole impactors Air-O-Cell and Bukard samplers – the slit impactors, on microscope slide or tape The Reuter centrifugal sampler (RCS) – portable – d50 ~ 3.8µ m The AGI-30 and the AGI-40 can only be used with water-based collection fluids The BioSampler can be used with nonevaporative liquids (mineral oil) – permit long sampling time 50
  51. 51. Bioaerosol Samplers: Collection Time Bioaerosol concentration vary greatly with time – sample collection time is essential t1 – t2 – low concentration t3 – t4 – high concentration Sampling time – sufficiently long Average concentration: Ca ts – starting time V = Qt tf – finish time N = CaQt Q – sampling flow rate N Ca Q The surface density: δ= = t A A δo optimal surface density 51 A - viewing area
  52. 52. Bioaerosol Samplers: Optimal Collection Time Optimal sampling time for solid surface sampler δo optimal surface density δ < < δ o Insufficiently loaded samples δ > > δ o overloaded samples Adjusting the sampling period to obtain optimal surface density δ A Optimal sampling time: t= Ca Q The optimal sampling time for a given bioaerosol concentration is different for each sampler – sampler’s flow rate and collection surface area 52
  53. 53. Bioaerosol Samplers: Optimal Collection Time Optimal collection time for impingers Impinger samples are not sensitive to overloading or under- sampling because the liquid sample can be either diluted or concentrated depending on the concentration of collected bioaerosol particles in the liquid. Evaporation of sampling liquid and reaerosolization of already collected particles limit the sampling time in most impingers 53
  54. 54. Bioaerosol Samplers: Optimal Collection TimePermissible sampling parameter ranges for less than 10% change in collection efficiency with the AGI-4 and AGI-30 impingers when operated at a sampling rate of 12.5 L/min 54
  55. 55. Bioaerosol Analysis 55
  56. 56. Intermediate Processing:• Manipulate samples to be compatible with detection methodology  Take into account liquid or solid surface collection techniques  Ex. - microscopy – sample on solid surface (i.e. filter)• If samples in liquid media – dilutions to achieve countable concentrations 56
  57. 57. Non-viability Assays: Microscopy – Brightfield (Light)• Limit of resolution => 0.2 µ m• Time-consuming, tedious, expensive Salmonella (DIC) Cryptosporidium (DIC) 57
  58. 58. Non-viability Assays: Microscopy – Electron Microscopy• Electronically magnified images• Electrons => short wavelengths• Magnification up to 2,000,000x Hepatitis A virus Influenza virus 58
  59. 59. Non-viability Assays: Flow Cytometry – Cell Sorting 59
  60. 60. Non-viability Assays: Molecular Detection• Polymerase Chain Reaction (PCR)• Genetic hybridization 60
  61. 61. Viability Assays: Bacteria and Fungi – Agar Culture • Non-selective: Plate Count Agar, R2A Agar • Selective: mFC Agar, EC Agar, Salmonella-Shigella Agar, Malt Extract Agar (MEA – fungi) R2A Agar Salmonella-Shigella Agar 61
  62. 62. Viability Assays: Virus – Cell Culture uninfected Late cytophathic effects: Cell degeneration Plaque formation Enlarged cells Nuclear inclusions 62
  63. 63. Further Characterization: Biochemical Analyses 63
  64. 64. Further Characterization: Antibiotic Resistance Microdilution Disk Diffusion 64
  65. 65. Further Characterization: Molecular Sequencing Sequence Info Dendrogram 65
  66. 66. Further Characterization: Molecular Characterization: Ribotyping 66
  67. 67. Part II: Bioaerosol Sampling:Demonstration & Hands-on Practice 67
  68. 68. Bioaerosol Sampling Illustration: Illustration of Bioaerosol Sampling Procedure (Impactor - culture method) Step 1: Agar preparation Step 2: Sampler flowrate calibration Step 3: Sample collection with a viable sampler Step 4: Sample transportation Step 5: Sample condition Step 6: Sample analysis 68
  69. 69. Pre-sampling – agar plate preparation: 69
  70. 70. Pre-sampling – agar plate preparation: 70
  71. 71. Pre-sampling – agar plate preparation: 71
  72. 72. Pre-sampling – agar plate preparation: 72
  73. 73. Pre-sampling- sampler flowrate calibration: 73
  74. 74. Pre-sampling- sampler flowrate calibration: 74
  75. 75. Taking Samples in the Field: 75
  76. 76. Taking Samples in the Field: 76
  77. 77. Taking Samples in the Field: 77
  78. 78. Taking Samples in the Field: 78
  79. 79. Post-Sampling – agar plate incubation: 79
  80. 80. Post-Sampling – agar plate reading: 80
  81. 81. Post-Sampling – agar plate reading: 81
  82. 82. Post-Sampling – agar plate reading: 82
  83. 83. Concentration Determination: Example #1• A bioaerosol sampling campaign was conducted at a ambient location in vicinity of a egg production farm. AGI-30 viable sampler was used to take total bacteria samples. The air flow rate of the AGI-30 was controlled at 12.5 l/min and the sampling duration was 30 min. After the field sampling, the samples were transported to the lab at 4 oC. 83
  84. 84. Concentration Determination: Example #1 – Cont. • Impinger fluid from each sample was transferred to a sterile tube and its volume determined. Impinger fluid samples, and/or dilutions in F-tab containing 0.1% Tween 80, were plated in duplicate on Trypticase Soy Agar (TSA) for growth of bacteria. The TSA plates were incubated at 37º C. Plates were checked daily for growth of colonies and moved to 4º C when colonies were of appropriate size for identification and counting. 84
  85. 85. Concentration Determination: Example #1 – Cont.• Lab results of an Impinger sample: Total Bacteria Total Impinger bacteria Recip. Bacteria/ml fluid vol. inCount 1 Count 2 Average Dilution Imp fluid (ml) Impinger 11 24 17.5 10 ? 16 ? • Bacteria/ml Imp fluid = average count * Recip. Dilution • Total bacteria in impinger = Bacteria/ml Imp fluid * Impinger fluid vol. 85
  86. 86. Concentration Determination: Example #1 – Cont. • Concentration calculation: N ???cfu C= = = ???cfu / m 3 Q * t 0.0125m 3 / min* 30 min N = total bioaerosol counts = ??? cfu Q = sampling flowrate = 12.5 l/min * 0.001 m3/l = 0.0125 m3/min t = sampling duration = 30 min. 86
  87. 87. Concentration Determination: Example #2• If a one-stage Anderson viable sampler with a no-selective R2A agar plate was used to take total bacteria samples in a residence home. The air flow rate of the sampler was controlled at 28.3 l/min and the sampling duration was 10 min. After the field sampling, the samples were transported to the lab at 4 oC. The agar plates were incubated for growth of colonies. It was observed that colonies counts of a plate was 82. What was the concentration of the total bacterial in the air of this home? 87
  88. 88. Concentration Determination: Example #2 – Cont. • Concentration calculation: N ???cfu C= = = ???cfu / m 3 Q * t 0.0283m 3 / min* 10 min N = total bioaerosol counts = ??? cfu Q = sampling flowrate = 28.3 l/min * 0.001 m3/l = 0.0283 m3/min t = sampling duration = 10 min. 88
  89. 89. References: •Baron, P.A. and K. Willeke. 2001. Aerosol Measurement:Principles, Techniques, and Applications, 2nd edition. JohnWiley & Sons, New York. •Hinds, W.C. 1999. Aerosol Technology: Properties, Behavior, and Measurement of Airborne Particles, 2nd edition. John Wiley & Sons, New York. •Cox, C.S. and C.M. Wathes. 1995. Bioaerosols Handbook.Lewis Publishers. Washington D.C. 89
  90. 90. Acknowledge:• Supported by a National Research Initiative grant from the National Institute of Food and Agriculture, Air Quality Program (No. 2007-55112-17856) 90
  91. 91. Hands-on Practice: sampler flow calibration/check 91