Pig oral fluids. Jeff Zimmerman Iowa State University

Transcription

1 Pig oral fluids Jeff Zimmerman Iowa State University

2 Iowa State University Vet Dx Lab 11,000 OF samples tested in ,517 in 2011 >52,000 in 2012

3 Overview of applications 1. Surveillance of endemic infectious agents using antibody- or PCR-based assays In the future OIE-listed diseases & other diseases impacting animal health and international trade 2. Detection/monitoring antibiotics 3. Non-specific population health indicators? Total immunoglobulin (IgM, IgA, IgG), α-amylase (Fuentes et al., 2011), acute-phase proteins, e.g., C reactive protein, haptoglobin (Gutierrez et al., 2009)

4 Terminology Why oral fluid? Why is oral fluid a good diagnostic specimen? Contains local and serum-derived pathogens and antibodies Cameron and Carmen, 2005

5 Use in human diagnostic medicine The agglutinating properties of several body fluids during Malta Fever. Pollaci and Ceralo Oral fluid antibodies used to detect HIV infection. J Clin Microbiol 24(5): /45 HIV seropositive individuals had detectable salivary antibodies to viral antigens Feasibility of a diagnostic test for HIV not requiring venipuncture?

6 Use in human diagnostic medicine NY STD clinics implement oral fluid testing = 40% increase in number tested March 2005 May ,058 oral fluid HIV tests 442 (0.27%) false positive rate Ann Emerg Med 53:151 Sensitivity ~99% Specificity 99.8%

7 Detection of infections in humans using oral fluids Prickett & Zimmerman, Patógeno Blanco diagnostico Citation Bordetella pertussis IgG Litt et al., 2006 Coronavirus virus (PCR) Wang et al., 2004 Cryptosporidium parvum IgG Moss et al., 2004 Cytomegalovirus IgG, IgA Hochman et al., 1998 Dengue virus IgG, IgM Cuzzubbo et al., 1998 Diptheria toxoid IgG, IgA Newcomb et al., 1969 Ebola virus IgG, PCR Formenty et el., 2006 Epstein-bar virus IgG, IgA Hochman et al., 1998 Hantaan virus IgG, IgM Petraityte et al., 2007 Helicobacter pylori IgG Luzza et al., 1995 Hepatitis A, B, C IgM Amado et al., 2006 Human herpes virus type 1 virus (PCR) Scott et al., 1997 Human herpes virus type 6 virus (PCR) Collot et al., 2002 Human leptospirosis IgM da Silva et al., 1992 Influenza virus IgA Waldman, 1968 Japanese encephalitis IgG, IgM, IgA Prasad et al., 1994 Sarampión IgM Brown et al., 1994 Paperas Anticuerpos neutralizantes Chiba and Nakao, 1972 Paperas IgM Frankova and Sixtova, 1987 Norwalk virus IgG, IgA Moe et al., 2004 Polio virus IgA Berger, et a., 1967 Rotavirus IgA Ward et al., 1992 Rubella IgG Ben Salah et al., 2003 Salmonella tyhpi IgA Herath, 2003 Treponema pallidum IgG Baguley et al., 2005 Trypanosoma cruzi IgG Pinho et al., 1999 Varicella-zoster virus virus (PCR) Furuta et al., 2004 Yersinia entercolitica IgG Grönblad and Mäkelä,1986

8 Oral fluid collection from pigs 1,2,3,4 1. Use cotton rope 2. Adjust length to pig size 3. Extract fluid from rope 4. Pour fluid into a tube chill or freeze 5. Submit to lab for testing ±75% of pigs in a pen of will interact with the rope in minutes

9 Why cotton material? Collection material can affect the results Material PRRS OF ELISA PRRS S/P (%+) PRRSV neutralizing antibodies PRRSV RT- PCR Cotton (n = 104) (45%) 1:11 34 (33%) Hemp (n = 104) (46%) 1:16 5 (5%) Nylon (n = 104) (45%) 1:30 2 2%)

10 Oral fluid collection from pigs 1. Use cotton rope 2. Adjust length to pig size 3. Extract fluid from rope 4. Pour fluid into a tube chill or freeze 5. Submit to lab for testing ±75% of pigs in a pen of will interact with the rope in minutes

11 70% of the pigs in a pen interact with the rope in 20 minutes Observations in 143 pen 4 barn on 3 sites Pigs 10- to 12-weeks of age Pens of 25 to 28 pigs

12 n = 143 pigs in 143 pens Understanding animal behavior is fundamental

13 Seddon et al Optimizing oral fluid collection from groups of pigs. Vet J (in press)

14 Pictures courtesy of Dr. Keith Erlandson

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17 Work with pig behavior Pigs explore the world in bites and chews. Train (non-threatening exploration), then collect Put the rope in cleanest part of the pen - away from drinkers and feeders Collect samples in the morning - when pigs most active

18 Oral fluids and PCR some examples

19 Classical swine fever virus Weesendorp et al Vet Microbiol 133:9-22

20 PCV2 Prickett et al Transboundary & Emerging Diseases

21 Influenza virus Detection of influenza virus by RT-PCR: nasal swabs vs. oral fluids in a wean-to-finish barn (Allerson and Torremorell, 2012)

22 PRRSV 3 trial, each with 24 boars Ingelvac MLV Type 1 field isolate Type 2 field isolate Kittawornrat et al., Virus Res 154:

23 Serum vs oral fluid: Area under the curve" correlation analysis (r = 0.68)

24 Serum vs. oral fluid - PRRSV PCRs Kittawornrat et al., Virus Res 154:

25 Potential for PCRs.? Foot-and-mouth disease virus African swine fever virus Classical swine fever virus Influenza viruses PRRS virus PCV2 TTV1 and 2 Erysipelothrix spp. M. hyorhinis, M. hyosynoviae, M. hyopneumoniae Others

26 but wait a minute

27 Effect 10 wean-finish of extraction sites protocol monitored using on PRRSV oral fluids PCR

28 Effect 10 wean-finish of extraction sites protocol monitored using on PRRSV oral fluids PCR

29 On-going improvements in PRRSV PCR Extraction Master Mix 1X (n = 63) Master Mix 2X (n = 63) A %* 27-43%* A %* 35-56%* A %* 24-38%* B %* 20-32%* B %* 21-33%* * Significantly different (p 0.05) from best protocol (McNemar s test) Chittick et al., J Vet Diagn Invest 23:

30 Performance of Influenza A virus PCRs 180 well-characterized samples Oral fluid spiked with H1N1 or H3N2 180 samples = (2 viruses x 8 dilutions x 10 samples per dilution) + 20 negatives Samples randomized then sent to 8 labs. 12 PCR protocols run

31 Probability of detecting H1N1 in oral fluid by Dilution (8 labs, 12 PCR protocols, 100 samples)

32 Probability of detecting H1N1 in oral fluid by Dilution (8 labs, 12 PCR protocols, 100 samples

33 Probability of detecting H3N2 in oral fluid by Dilution (8 labs, 12 PCR protocols, 100 samples

34 RT-PCR and sequencing: Comments on work in progress

35 Mucin monomer Gut Miocrobes 1:4, Model of 5 mucin monomers. Mucus gels are composed of monomers. Mucosal Immunology (3rd ed), p. 58

36 Mucin 1. Present in oral fluid (all species) 2. Aggregation is time-dependent. - Explains why PCR/sequence success by time? 3. Mucin binds and/or inactivates pathogens - HIV, rotavirus, influenza virus, norovirus, coronavirus, mycoplasma, bacteria 4. Antibody is associated with mucin aggregates, but easily passes through. 5. Treatments to reverse mucin aggregation?

37 Take Home - PCRs 1. PCRs are not created equal Which PCR is your lab running? 2. More PCR optimization necessary 3. Oral fluids contain something probably mucin that affects PCRs and sequencing

38 Oral fluids & antibodies some examples

39 Antibody in oral fluids Antibody assays are cheaper than PCRs Widely used in human oral fluid testing Most serum antibody assays can be optimized to detect antibodies in oral fluid (Cameron and Carmen, 2005) Change incubation time, sample dilution, reagent dilutions, and adjust cut-off value

40 Anti-PCV2 IgG (L) and IgA (R) in oral fluids Prickett et al Transboundary & Emerging Diseases

41 Antibody against influenza A virus

42 PRRSV IgM and IgG in oral fluids Luminex (microsphere) assay Dr. Bob Rowland, Kansas State University

43 Development of an oral fluid antibody test 1. Adaptation of PRRS X3 serum ELISA 2. Response in Reference standards Experimental samples Field samples 3. Test performance estimates

44 Adaption of the PRRS X3 serum ELISA Serum Sample dilution: 1:40 Sample volume: 100 μl Incubation time: 30 min Kit conjugate Oral fluid Sample dilution: 1:2 Sample volume: 250 μl Incubation time: 16 hours Titrate conjugate

45 SAMPLE SET #1 (reference standards) 1,100 head wean-to-finish barn Vaccinated BIVI PRRS MLV on Day 0 Samples collected DPV -1, 10, 15, 20, 28, 35, 41, 49, 56, 75, 91 (n = 11)

46 Reference standards DPV -1, 10, 15, 20, 28, 35, 41, 49, 56, 75, 91

47 SAMPLE SET #2 (experimental - Bob Rowland) 7 trials 200 pigs per trial PRRSV isolate NVSL , GenBank accession number AY545985

48 SAMPLE SET #3 (field samples n = 600) Wean-to-finish barns (1100 pigs) 10 sites x 6 pens per site x sampling every 2 weeks for 18 weeks.

49 Response in field samples (sub-sample) WEEK POST PLACEMENT Barn Pen P P P NS P P P P P P P P 1.46 P P P 2.42 P P

50 Neg ELISA Pos ELISA Pos ELISA PCR P Pos PCR WEEK POST PLACEMENT Barn Pen P P P NS P P P P P P P P 1.46 P P P 2.42 P P

51 Neg ELISA Pos ELISA Pos ELISA PCR P Pos PCR WEEK POST PLACEMENT Barn Pen P P P NS P P P P P P P P 1.46 P P P 2.42 P P

52 Neg ELISA Pos ELISA Pos ELISA PCR P Pos PCR WEEK POST PLACEMENT Barn Pen P P P NS P P P P P P P P 1.46 P P P 2.42 P P

53 Neg ELISA Pos ELISA Pos ELISA PCR P Pos PCR WEEK POST PLACEMENT Barn Pen P P P NS P P P P P P P P 1.46 P P P 2.42 P P

54 Neg ELISA Pos ELISA Pos ELISA PCR P Pos PCR WEEK POST PLACEMENT Barn Pen P P P NS P P P P P P P P 1.46 P P P 2.42 P P

55 Neg ELISA Pos ELISA Pos ELISA PCR P Pos PCR WEEK POST PLACEMENT Barn Pen P P P NS P P P P P P P P 1.46 P P P 2.42 P P

56 Neg ELISA Pos ELISA Pos ELISA PCR P Pos PCR WEEK POST PLACEMENT Barn Pen P P P NS P P P P P P P P 1.46 P P P 2.42 P P

57 Neg ELISA Pos ELISA Pos ELISA PCR P Pos PCR WEEK POST PLACEMENT Barn Pen P P P NS P P P P P P P P 1.46 P P P 2.42 P P

58 Neg ELISA Pos ELISA Pos ELISA PCR P Pos PCR WEEK POST PLACEMENT Barn Pen P P P NS P P P P P P P P 1.46 P P P 2.42 P P

59 Neg ELISA Pos ELISA Pos ELISA PCR P Pos PCR WEEK POST PLACEMENT Barn Pen P P P NS P P P P P P P P 1.46 P P P 2.42 P P

60 ELISA Negative ELISA Positive ELISA, PCR positive P PCR positive WEEK POST PLACEMENT Barn Pen P 1.19 P P P P 1.62 P 1.55 P P P 0.94 P 1.99 P 3.62 P P P 1.79 P P P 1.24 P P

61 Diagnostic sensitivity and specificity Sample sets #1, 2, 3 NEGATIVE SAMPLES Experimental = 84 Field neg (ISU VDL) = 283 TOTAL = 367 POSITIVE SAMPLES Experimental = 253 Field = 241 TOTAL = 494

62 Sensitivity / Specificity Establishing a cutoff

63 S/P Ratio Sensitivity (%) (95% CI) Specificity (%) (95% CI) (93.1, 97.0) 99.2 (97.6, 99.8) (93.1, 97.0) 99.5 (98.0, 99.9) (93.1, 97.0) 99.7 (98.5, 100.0) (92.9, 96.9) (99.0, 100.0) (92.4, 96.5) (99.0, 100.0)

64 Potential for antibody assay.? Foot-and-mouth disease virus African swine fever virus Classical swine fever virus Influenza viruses PRRS virus PCV2 Aujeszky's disease Erysipelothrix spp. M. hyorhinis, M. hyosynoviae, M. hyopneumoniae APP, Salmonella, others

65 Fast, easy, cheap surveillance using PCRs using antibody assays

66 LEVEL TWO Bits of things

67 Oral fluid sample: What kind of sample is it?

68 Is a pen-based sample a pooled sample? A pooled sample is created by combining 2 specimens into one aliquot for testing.

69 Is a pen-based sample a pooled sample? A pooled sample is created by combining 2 specimens into one aliquot for testing. First dx application Dorfman, Pooling always results in loss of information

70 A pen-based sample is a grab sample A single sample collected at a particular time and place that represents the composition of the water, air, or soil only at that time and place. Apalachicola Bay, Florida

71 A pen-based sample is a grab sample Place, time, and sample unit data? "Yes" for grab samples; often "No" for pools "Yes" allows for statistical analysis of the results Apalachicola Bay, Florida

72 Oral fluid samples: Effect of collection material, processing and sampling order on assay results

73 Material, collection order & processing Olsen et al. Res Vet Sci (submitted) OF samples collected from 104 pens 3 types of rope - cotton, hemp, nylon Pens sampled in 1 of 3 sequences C-N-H; N-H-C; or H-C-N. Samples processed or unprocessed 10,000 x g for 2 hours and then filtered using a 0.22µm

74 Factors Material (univariate analysis) Total Mean Antibody (mg/ml) IgM IgA IgG Cotton (n = 104) b c a Hemp (n = 104) a b a Nylon (n = 104) a a a Processed 1 (univariate analysis) NO (n = 312) a a a YES (n = 312) b b b Collection Order (univariate analysis) 1 (n = 104) a a a 2 (n = 104) b b b 3 (n = 104) c b b 1 Centrifuged at 10,000 x g for 2 hours and then filtered (0.22µm)

75 Factors Material (univariate analysis) Total Mean Antibody (mg/ml) IgM IgA IgG Cotton (n = 104) b c a Hemp (n = 104) a b a Nylon (n = 104) a a a Processed 1 (univariate analysis) NO (n = 312) a a a YES (n = 312) b b b Collection Order (univariate analysis) 1 (n = 104) a a a 2 (n = 104) b b b 3 (n = 104) c b b 1 Centrifuged at 10,000 x g for 2 hours and then filtered (0.22µm)

76 PRRSV Assays Factors PRRSV IgG ELISA VN Antibody-1 2 VN Antibody-2 2 qrt-pcr (%) Material Cotton (n = 104) a (45%) 1:12.4 b 1:10.1 b 34 a (32.7%) Hemp (n = 104) a (46%) 1:17.9 ab 1:14.1 b 5 b (4.8%) Nylon (n = 104) a (45%) 1:27.2 a 1:32.0 a 2 b (1.9%) Processed 1 NO (n = 312) a (46%) nt nt 41 (13.1%) YES (n = 312) b (45%) 1:18.1 1:16.6 nt Collection Order 1 (n = 104) a (45%) 1:21.3 a 1:19.2 a 16 a (15.4%) 2 (n = 104) a (46%) 1:17.8 b 1:16.2 b 16 a (15.4%) 3 (n = 104) b (45%) 1:15.8 b 1:14.8 b 9 a (8.7%) 1 Centrifuged at 10,000 x g for 2 hours and then filtered (0.22µm) 2 VN antibody titers based on type 2 isolates VR-2332 (1) or MN-184 (2)

77 Material, collection order & processing Olsen et al., Res Vet Sci (submitted) Conclusions: Any of the three factors could affect results, but the effects were non-uniform and assay-dependent. Keep the analyte in mind when selecting collection material Cotton is the best choice for PRRSV assays (ELISA or RT-PCR)

78 Oral fluid samples: Effect of prevalence on detection

79 Experimental Design Olsen et al., (in progress) 2012 Objective: Estimate probability of detecting PRRSV infection by ELISA or PCR using a penbased oral fluid sample Design 25 pens of 25 pigs 5 treatment groups (0, 1, 3, 5, 9 MLV vaccinated pigs), 5 pens per treatment

80 Experimental Design (Timeline) Dec 23 Jan 4 Vaccinate pigs with MLV PRRSV (off-site) Collect one oral fluid sample from each pen in study (negative controls) Jan 5 (am) Bleed 535 pigs in 25 pens (negative controls) Jan 5 (pm) Introduce 90 vaccinated pigs into trt pens Jan 6 (am) Collect 5 successive oral fluid samples from each of 25 pens while monitoring the time each viremic pig in the pen chewed the cotton collection rope Jan 6 (am) Bleed 90 viremic pigs after collecting oral fluids

81 Probability of detection as a function of prevalence

82 Testing PRRSV RT-PCR - six diagnostic labs 90 serum samples from vaccinated animals 150 oral fluid samples (25 pens x 6 collections) 535 "background pig" serum samples pooled by 5s - 2 labs PRRSV ELISA - six diagnostic labs 90 serum samples from vaccinated animals 150 oral fluid samples (25 pens x 6 collections) 535 "background pig" serum samples - 2 labs

83 Probability of detection PRELIMINARY ANALYSIS PRRSV RT-PCR PRRSV ELISA Probability of detection if one randomly selected pig were bled and tested (ELISA or RT-PCR), assuming 100% sensitive and specific assays Within-pen prevalence (%)

84 Oral fluid samples: Sample size and frequency (work in progress)

85 Sample size and frequency Wang et al. Design and analysis of PRRSV surveillance: Temporal and spatial sampling, mapping, monitoring and automated rapid detection of outbreaks. NPB Will combine Wang's statistical theory with results from the field

86 PRRS oral fluid antibody assays: Exogenous sources of PRRSV antibody (Contribution from Dr. JK Johnson)

87 2500 hd Sow Farm produces 1000 hd weekly PRRS negative system Nursery Site 1 Nursery Site 2 Barn A 1000 hd Barn B 1000hd Barn A 1000 hd Barn B 1000hd Barn C 1000 hd Barn D 1000 hd Barn C 1000 hd Barn D 1000 hd Finishing Site Five 1000 hd Barns Finishing Site Five 1000 hd Barns

88 Routine nursery PRRS surveillance 12/12/2011 SITE LT* PRRS PCR PRRS OF ELISA (S/P) LT* PCR retest PCR follow-up Tetracore PCR Gelbased PCR Barn 1 neg neg neg neg Barn 2 neg neg neg neg Barn 3 neg neg neg neg Barn 4 neg neg neg neg *Life Technologies Corp

89 Routine nursery PRRS surveillance 12/12/2011 SITE LT* PRRS PCR PRRS OF ELISA (S/P) LT* PCR retest PCR follow-up Tetracore PCR Gelbased PCR Barn 1 neg neg neg neg Barn 2 neg neg neg neg Barn 3 neg neg neg neg Barn 4 neg neg neg neg *Life Technologies Corp

90 91 oral fluids from nursery 12/16/2011 SITE LT* PRRS PCR PRRS oral fluid ELISA (mean S/P) ISU BIVI HMC Barn 1 23 neg 21 neg (0.09) 21 neg (0.13) Barn 2 24 neg 23 pos (0.94) 23 pos (1.10) Barn 3 23 neg 24 pos (3.64) 24 pos (3.42) Barn 4 21 neg 21 pos (1.84) 23 pos (1.72) *Life Technologies Corp

91 40 serum samples from nursery barn 3 12/20/2011 Samples 40 serum samples - 8 pools of 5 for PRRS PCR - Tested individually on PRRS ELISA LT PRRS PCR All negative PRRS X3 serum ELISA All negative Mean S/P = 0.01 Range

92 PRRS antibody in transition diet 12/28/2011 PRRS oral fluid ELISA (mean S/P) Sample - Feed sample representative of barns 2, 3, & 4-5 g feed + 20 ml diluent. - Diluent tested for PRRSV antibody Sample dilution 1:2 1:4 1:8 1:16 1:32 1:64 1:128 1:256 1:512 ISU BIVI HMC Feed tested on the suggestion of Drs. Bill Dubois and Scanlon Daniels

93 Note that goes out with ISU VDL PRRS X3 oral fluid results: Caution: The PRRS X3 oral fluid antibody assay may detect antibodies against PRRSV in samples collected from pigs consuming diets containing spray-dried plasma of porcine origin.

94 Gracias!