Universidad de Las Palmas de Gran Canaria, Arucas, Spain, 2
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1 Efficacy of Sodium Dodecyl Sulfate and Formic acid inactivation of Caprine Arthritis Encephalitis virus in vitro A. Morales delanuez 1, P. Plummer 2, S. Hartmann 3, P. Nara 4, A. Argüello 1, J. Trujillo 4 ; 1 Universidad de Las Palmas de Gran Canaria, Arucas, Spain, 2 ISU VMPM, Ames, IA, USA, 3 Drexel University, Philadelphia, PA, USA, 4 ISU CAHDIT, Ames, IA, USA. Jessie Trujillo jtrujill@iastate.edu
2 0/09/cae virus el origen.html Background CAE is considered one of the most devastating diseases of dairy goats(turin at el., 2005). In the USA, the prevalence of serum antibodies against CAEV in dairy goat herds ranges from 38 to 81% (Concha Bermejillo, A. de la, 2003) Caprine arthritis encephalitis Virus produces a disease characterized by chronic proliferative synovitis and periarthritis and acute afebrile leukoencephalomyelitis (Al Ani and Vestweber, 1984) Further studies have demonstrated that transmission can indeed occur via ingestion of infected colostrum (Cork et al., 1974) (Adams et al., 1983)
3 Background Colostrum is a nutrient rich fluid produced by female mammals immediately after giving birth. Due to its high content in Ig, mainly IgG, colostrum provides confers passive immunity in the newborn (Foley and Otterby, 1978). Pasteurization can play a vital role in preventing such transmission diseases like CAE. Several authors have confirmed this like Adams et al. (1983)
4 Nutrition Immunity Intestine Motility Introduction acterias aerobias diferentes_ htm icles/ htm
5 Colostrum Treatments Nutrition Immunity Energy Intestine Motility Introduction acterias aerobias diferentes_ htm icles/ htm
6 Background Finnish farmers have ten years of practical experience with free access feeding of milk acidified with formic acid to preserve it for one to three days Mix 40 to 45 ml of dilute acid (8.5%) into one litre of colostrum and test that the ph is down than 4.5 ( Formic acid treated colostrum is not statistically different than non treated colostrum in immunoglobulin concentration or functionality and can be fed without consequence to passive transfer from dam to calf (Quirk et al. 2011)
7 lfato_s%c3%b3dico Background Treating the milk of HIV 1 positive mothers with relatively low concentrations SDS allowed for safe breastfeeding (Hartman et al., 2006) Treatment of goat colostrum with 1% SDS reduced the bacterial load to a degree similar to that seen with pasteurization The IgG in SDS treated colostrum does not effect IgG concentration or biological function Goat kids fed SDS treated colostrum have not difference in biological parameters and showed normal immune passive transfer (Morales delanuez etal.,2011)
8 In vitro Inactivation of CAEV in goat colostrum Preliminary inactivation studies used DMEM spiked with known virus titer Assay endpoint: virus titer reduction assay on goat synovial membrane cells (GSM) in duplicate; in triplicate non treatment controls in colostrum and DMEM; Percent inactivation based on like controls
9 CAEV Strains CAEV CO: Pathogenic molecular clone constructed from the provirus (brain) from a goat with encephalitis; moderate passage in GSM (1990) CAEV 63: Pathogenic biological clone originally isolated from a goat with arthritis; high passage in GSM ( ) CAEV 1g5: More recent pathogenic isolate from a goat with arthritis; PBMC co culture; low passage in GSM (1998)
10 Colostrum samples All from CAEV sero negative goats Colostrum 1: Long term cryostorage ( 6 plus years) Colostrum 2: pooled from 2 3 goats fresh frozen (<8 months) Colostrum 3: pooled from 3 goats fresh frozen All tested as skim colostrum; cell pellet and fat plug removed.
11 DMEM/ colostrum SDS/ Formic acid 1%/ ph3 0.1% ph4 ph corrected; partial SDs removal before titration assay CAEV solutions.com/custom Lentivirus Reporter GenesiRNA_p_64.html 0.01% ph % ph7 10% SDS Or acidification Control W/O SDS or acid Material and Methods
12 Virus titration assay: Reed and Munch Goat Synovial Membrane cells Primary cells Material and Methods Serial Dilutions Incubation to 37ºC + 5% CO 2, 14 days Change the media (DMEM) day 7 of incubation D14 fixed and stained and scored 50% end point calculated
13 Tissue Culture Infection Dose 50 (TCID 50 ) Material and Methods
14 Hypotheses We hypothesize that Formic Acid treatment of colostrum will inactivate CAEV AIM 1) Determine the efficacy of various concentrations of Formic Ac. in inactivation of 3 CAEV strains in the presence colostrum. We hypothesize that SDS treatment of colostrum will inactivate CAEV AIM 1) Determine the efficacy of various concentrations of SDS in inactivation of 3 CAEV strains in the presence of colostrum.
15 Formic Acid
16 Formic Acid. ph vs Time ph3 ph4 ph5 ph7 Red. 15 ND ND 5.57± ± % 30 ND ND 5.67± ± % or 100% 1h ND ND 6.11± ± % 2h ND ND 5.49± ± % 4h ND ND 5.51± ± % Data reported as log TCID 50 ND not detected Assay LOD
17 Inactivation of CAEV CO achieved at various at levels of acidification in colostrum using formic acid CAEV CO Colostrum Time ph 3 ph 4 ph 4.5 contro l Amount Virus Added Percent Reduction* ph 4 SEM 1 15 N.D a N.D a % N.D a N.D a % N.D a N.D a N.D a % N.D a N.D a N.D a % N.D a N.D a 2.82 b % N.D a N.D a 2.84 b % 0.32 a=assay LOD 10 1 TCID 50
18 Inactivation of CAEV 63 achieved at various at levels of acidification in colostrum using formic acid CAEV 63 Percent Reduction* Colostru m Time ph 3 ph 4 ph 4.5 Control Amount Virus Added ph 4 SEM N.D a N.D a 3.84 b % N.D a N.D a 3.92 b % N.D a N.D a N.D a % N.D a N.D a N.D a % N.D a N.D a N.D a % N.D a N.D a N.D a % 0.43 a=assay LOD 10 1 TCID 50
19 Inactivation of CAEV 1g5 achieved at various at levels of acidification in colostrum using formic acid CAEV 1g5 Percent Reduction* Colostrum Time ph 3 ph 4 ph 4.5 Control Amount Virus Added ph 4 SEM N.D a N.D a % N.D a N.D a % N.D N.D N.D N.D N.D a N.D a N.D a % N.D N.D N.D N.D N.D N.D N.D N.D a= assay LOD 10 1 TCID 50
20 SDS inactivation of CAEV CO in DMEM TCID50 1% 0.1% 0.01% 0.001% Positive 1ºtime ªTime Media Red Log Data reported as log TCID 50 Red % 99.76% 99.95%
21 SDS
22 Inactivation of CAEV CO with two concentrations of Sodium Dodecyl Sulfate in colostrum CAEV CO Percent Reduction Colostrum 1%SD S 0.1%SDS No SDS Amount Virus Added 1 %SDS SEM 0.1%S DS SEM 1 N.D % % N.D N.D % N.D % % 0.22 Reported percent reduction are minimums and could be greater since assay detection limits for TCID 50 for colostrum 1, 2 and 3 are 2.35, 2.78 and 2.18 TCID 50, due to the effects of concentrated SDS on GSM cells uses in the assay.
23 Inactivation of CAEV 1g5 with two different concentrations of SDS in goat colostrum. CAEV 1g5 Percent Reduction* Colostru m 1%SDS 0.1%SDS No SDS Amount Virus Added 1%SDS SEM 0.1%SDS SEM 1 N.D N.D % % N.D N.D N.D N.D N.D 3.73 *Reported percent reduction are minimums and could be greater since assay detection limits for TCID 50 for colostrum 1, 2 and 3 are 1.0, 2.45 and 2.54 TCID 50, due to the effects of concentrated SDS on GSM cells uses in the assay.
24 Why are CAEV titers so low with fresh frozen colostrum? Fresh frozen colostrum pools (colostrum 2 and 3) Natural antimicrobial components of colostrum Immunoglobulins Complement, interferon, Chitotriosidase Lactoferrin Other bioactive components such as insulin like growth factors, lysosyme, lactoperoxidase and many others
25 Natural Inactivation of various CAEV strains in fresh frozen goat colostrum (colostrum 2 and 3) compared to long term cryopreserved colostrum 1 Colostrum Colostrum Added Amount Virus Added Percent Reduction* CAEV CO CAEV 1g5 CAEV NONE % NONE NONE % 3 N.D % NONE % %
26 Conclusions Both Formic acid acidification to a ph of 4 and SDS treatment at 1% are effective in partial to complete inactivation of CAEV in colostrum Both formic acid and SDS treatment of milk and colostrum have been shown to not affect Passive transfer or kid or calf health Fresh goat colostrum has significant natural ability to in activate CAEV; this activity is dependent on colostrum and viral factors
27 Next step Evaluate in vivo efficacy of Formic Acid and SDS in the prevention of CAEV infection following oral challenge To be performed spring summer 2013 Funded by the American Dairy Goat Association Research Foundation (2013)
28 Thanks for listening! Thanks to the Dairy goat Producers in Iowa for samples Spanish government and ISU/CVM/CAHDIT for funding All the folks and the goats: A. Morales delanuez P. Plummer S. Hartmann P. Nara A. Argüello Heartland Dairy MO in vivo Jessie Trujillo
29 Characterization of CAEV in USA CO g5
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