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Title: 1
Thermal Inactivation of Infectious Pancreatic Necrosis Virus (IPNV) in a Peptone-Salt 2
Medium Mimicking the Water-soluble Phase of Hydrolyzed Fish By-Products 3
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Running title: 5
Thermal Inactivation of IPNV 6
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Authors: 8
Halvor Nygaard 1*, Ingebjørg Modahl 2 and Mette Myrmel 2 9
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Addresses: 11
Nofima AS, Fyllingsdalen, Norway 1, National Veterinary Institute, Oslo, Norway 2 12
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Key words: 14
IPNV, inactivation, fish by-products 15
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* Corresponding author: 18
Mailing address: Nofima AS, Kjerreidviken 16, 5141 Fyllingsdalen, Norway. Phone: 19
(+47) 55501233. Fax: (+47) 55501299. E-mail: [email protected]. 20
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Copyright © 2012, American Society for Microbiology. All Rights Reserved.Appl. Environ. Microbiol. doi:10.1128/AEM.07358-11 AEM Accepts, published online ahead of print on 13 January 2012
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ABSTRACT 27
IPNV (serotype Sp) was exposed to temperatures from 60 to 90ºC in a medium 28
mimicking the water soluble phase of hydrolyzed fish by-products. D-values ranged 29
from 290 to 0.5 min and the z-value was approximately 9.8ºC. Addition of formic acid 30
to pH 4 did not enhance heat inactivation. Predicted inactivation effects at different 31
temperature-time combinations are provided. 32
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34
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In 2008, the global capture and aquaculture production of fish was 90 and 53 million 36
tonnes, respectively. Of this production, 19 % was destined for fishmeal and -oil 37
production (6, 7). Most known fish pathogens have been detected in wild fish (15). 38
Both wild and farmed fish can represent a risk for transmission. The Animal By-39
Products Regulation (EC) no 1069/2009 categorizes fish by-products according to 40
the level of risk they represent to public and animal health (13, 14). Category 3 by-41
products are whole wild fish caught for the purpose of fishmeal production and fresh 42
by-products from wild and farmed fish caught for human consumption. These 43
products are allowed for manufacturing of feed. Fishmeal must be submitted to a 44
processing method which ensures that the product complies with microbiological 45
standards (13, 14). Category 2 by-products may include dead fish from disease 46
outbreaks. Steam pressure sterilization is required before the material can be 47
disposed as waste, or transformed into biogas or organic fertilizers. IPNV is regarded 48
as one of the most heat resistant fish-pathogens (3, 4); however, few kinetic data on 49
thermal inactivation are available. 50
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This study presents D-values and z-values for heat inactivation of IPNV in a relevant 52
matrix. Such data are needed by the industry and authorities in order to decide 53
whether thermal processes provide adequate inactivation of fish pathogens. As 54
thermal inactivation may be affected by the heating medium, natural matrices should 55
be used in experiments. High content of organic matter has been shown to protect 56
viruses (1). However, as salmon by-products exhibited an adverse toxic effect on 57
the BF-2 cells, fish homogenate was substituted by a peptone-salt-medium (PSM) 58
containing peptone (121 g/l) and NaCl (2 g/l). The pH was adjusted to 7.0 with NaOH 59
or to 4.0 with formic acid. The PSM contained 10.2 % protein and 0.3 % salt which 60
are similar to the water phase of fish silage and therefore more relevant than cell 61
culture media used in previous studies. 62
63
IPNV (serotype Sp) was grown on BF-2 cells at 15°C using EMEM with 10 % FBS, 4 64
mM L-glutamine and 50 µg/ml Gentamicin. After 7 days, virus was harvested by 65
freeze/thawing and kept at 4°C. IPNV was titrated on cells (without FBS) in 96 well 66
plates with 6 parallels. Virus suspensions were left on the cells for two hours, 67
medium was added and after 7 days, the cells were screened for cytopathogenic 68
effect. The virus titre was determined by the Kärber method (9). The detection limit of 69
the assay was 2.8 Log10 TCID50/ml. IPNV suspension with titre 6.6 ± 0.2 Log10 70
TCID50/ml was diluted 1:10 in PSM (pH 7 and 4) and submerged (2.2 ml in 14 x 100 71
mm glass tubes) in water baths at temperatures between 60 and 90ºC. The heat 72
treatment was terminated by cooling in ice/water. The temperature in PSM during 73
heating was less than 1°C under target temperatures after 60 seconds. 74
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The heat resistance of micro-organisms are frequently described by D-values (time 76
needed to kill 90 % of a population at a certain temperature) and the z-value 77
(temperature change needed to alter the D-value by a factor of 10). Plotting the 78
logarithmic number of survivors versus time, provide inactivation curves where the D-79
value is the negative reciprocal of its slope. Similarly, the z-values are calculated 80
from a plot of Log D-values versus exposure temperatures. The relationship between 81
D-values and temperature is expressed by Bigelow’s equation (2): 82
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Log Dx = (Ty – Tx)/z + Log Dy 84
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where Dx and Dy are decimal reduction times at temperature Tx and Ty. With 86
Bigelow’s equation, D-values can be estimated for other temperatures than those 87
experimentally examined. 88
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A pre-study determined the appropriate combinations of exposure times and 90
temperatures and indicated that IPNV was more stable at pH 4 than at pH 7 (Table 91
1). The inactivation curves at 60, 70, 80 and 90ºC fitted first order kinetics, except at 92
60ºC where approximately 80 % of infectivity was lost during the first min (Fig. 1). D-93
values obtained at pH 4 and 7 are quite similar (Table 2). However, the amount of 94
infective IPNV is at least 1 log10 higher at pH 4 than pH 7, even before heat treatment 95
(Fig. 1). The effect of pH could be due to conformation change of viral proteins at 96
higher pH which reduces virus infectivity. As IPNV seems to be less stable at pH 7, 97
studies should be performed on the effect of alkaline storage. 98
Basis for z-value calculation was D-values at 60, 70 and 80ºC. At 90ºC IPNV was 99
reduced to below detection limit before the target temperature was reached. The z-100
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value for pH 4 and 7 was 9.8ºC (R2 value= 0.97) and 9.9ºC (R2 value= 0.98), 101
respectively. Plots of Log D-values versus exposure temperatures showed that the 102
temperature must be approximately 1.6ºC higher at pH 4 than pH 7 to achieve equal 103
inactivation. 104
105
Previous studies have demonstrated that thermal inactivation of IPNV is biphasic. A 3 106
Log10 reduction was found at 60ºC during the first 30 min and then 1 Log10 reduction 107
per 80 min (10). Similarly, 60ºC gave a 1.5 Log10 reduction after 20 min and then 1 108
Log10 reduction per 160 min (8). DEFRA (3) studied inactivation of IPNV at 60ºC and 109
found that serotype Sp was reduced by 5.1 Log10 in 24 hours. Smail et al. (12) found 110
that 5 hours at 60ºC gave a 2.58 Log10 reduction of strain Sp in fish silage. The titre 111
decreased by 2 Log10 the first 2 hours. With an initial concentration of 2-2.5 Log10 112
pfu/ml, which was claimed to be typical for native silages, no infective virus was 113
detected after 1 hour. The conclusion was that 2 hours treatment of fish silage at 114
60ºC would be sufficient for virus inactivation, allowing the silage to be used for 115
feedstuff. The inactivation curve at 60ºC at pH 4 in our study seems to correspond 116
well to the results of Smail et al. For 70, 80 and 90ºC there is limited information on 117
D-values. In a study on IPNV (VR-299), 8 hours, 2 hours and 10 min were needed to 118
reduce infectivity to below the detection limit at 60, 70 and 80ºC, respectively (16). 119
This is consistent with our results. In the same investigation, it was found that IPNV 120
decreased more rapidly when heated to 65ºC at neutral pH than in pH 4. 121
122
A Norwegian regulation laid down in 2007, required a minimum of 3 Log10 reduction 123
of infectious IPNV in materials from aquaculture fish used as feed for aquaculture 124
fish. The regulation was repealed after the former Animal By-Products Regulation 125
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(EC) no 1774/2002 came into force, but is still a relevant reference to sufficient 126
inactivation of fish pathogens. 127
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In a traditional fishmeal process, category 3 by-products are heated to above 90ºC 129
and kept above 80ºC for 20 min. According to our estimates, 3 Log10 reductions of 130
IPNV will be achieved after 0.7 min at 90ºC, 7.1 min at 80ºC or 23 min at 75ºC (Table 131
3). Category 2 by-products have to be heat treated with e.g. steam pressure 132
sterilisation at 133ºC and 3 bar pressure for 20 min (13, 14). Our estimated effect of 133
this method was 106 Log10 reductions of IPNV. Therefore, a traditional fishmeal 134
process (5) provides adequate IPNV inactivation, while team pressure sterilization is 135
excessive in relation to inactivation of IPNV. 136
137
The Norwegian Seafood Federation has applied for an approval of an alternative 138
processing method for category 2 fish by-products: ensilage at pH below 4 for 24 139
hours before heat treatment at 85ºC for 25 min. EFSA has concluded that risks 140
related to pathogens present would be adequately reduced by the proposed method. 141
The final approval of the method is still pending. 142
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This work was supported by RUBIN, a foundation working for increased and 145
profitable utilization of by-products from fisheries and fish farming in Norway. We 146
thank Jørgen Seliussen, Hordafor AS for providing information on the composition of 147
fish silage and Gunn Harriet Knutsen, Norwegian Seafood Federation for critical 148
review of the manuscript. 149
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REFERENCES 151
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13. The European Commission. 2011. Commission Regulation (EU) No 142/2011 200
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TABLE 1. Log10 IPNV titer (TCID50/ml) after heat treatment in PSM 226
pH in medium Heat treatment
No heatinga 70ºC/25 min 85ºC/25 min
4.0 6.1 5.1 < 2.8
7.0 4.5 3.8 < 2.8 a: 20-25ºC/25 min 227
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TABLE 2. D-values for IPNV in PSM 230
Temperature pH 7.0 pH 4.0
D-value (min) R2 valuea D-value (min) R2 value
60ºC 287.82 0.35 291.47 0.39
70ºC 15.86 0.83 53.92 0.88
80ºC 2.74 0.74 2.65 0.96
90ºC 0.48 1.00 1.17 0.88a R2: Squared Correlation Coefficient 231
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TABLE 3. Estimated time (min) to inactivate IPNV in PSM 233
Inactivation Temperature
60ºC 65ºC 70ºC 75ºC 80ºC 85ºC 90ºC
1 Log10 reductions 102 32 10 3.0 0.9 0.3 0.1
2 Log10 reductions 441 137 42 13 4.0 1.2 0.4
3 Log10 reductions 781 242 75 23 7.1 2.2 0.7
4 Log10 reductions 1120 347 107 33 10 3.1 1.0
5 Log10 reductions 1460 452 140 43 13 4.1 1.3
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Figure legends 236
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FIG. 1. Inactivation curves for IPNV in PSM at pH 4.0 (open squares) and pH 7.0 238
(closed squares) at 60ºC (a), 70ºC (b), 80ºC (c) and 90ºC (d). The straight lines are 239
regression exponential curves. The data points at pH 4 are average of two parallell 240
analyses. Error bars represent standard deviation. 241
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