Cottingim et al. Porcine Health Management (2017) 3:5
DOI 10.1186/s40813-016-0048-8
RESEARCH
Open Access
Feed additives decrease survival of delta
coronavirus in nursery pig diets
Katie M. Cottingim1, Harsha Verma2, Pedro E. Urriola1, Fernando Sampedro2, Gerald C. Shurson1
and Sagar M. Goyal2*
Abstract
Background: Feed contaminated with feces from infected pigs is believed to be a potential route of transmission
of porcine delta coronavirus (PDCoV). The objective of this study was to determine if the addition of commercial
feed additives (e.i., acids, salt and sugar) to swine feed can be an effective strategy to inactive PDCoV.
Results: Six commercial feed acids (UltraAcid P, Activate DA, KEMGEST, Acid Booster, Luprosil, and Amasil), salt, and
sugar were evaluated. The acids were added at the recommended concentrations to 5 g aliquots of complete
feed, which were also inoculated with 1 mL of PDCoV and incubated for 0, 7, 14, 21, 28, and 35 days. In another
experiment, double the recommended concentrations of these additives were also added to the feed samples and
incubated for 0, 1, 3, 7, and 10 days. All samples were stored at room temperature (~25 °C) followed by removal of
aliquots at 0, 7, 14, 21, 28, and 35 days. Any surviving virus was eluted in a buffer solution and then titrated in
swine testicular cells. Feed samples without any additive were used as controls. Both Weibull and log-linear kinetic
models were used to analyze virus survival curves. The presence of a tail in the virus inactivation curves indicated
deviations from the linear behavior and hence, the Weibull model was chosen for characterizing the inactivation
responses due to the better fit. At recommended concentrations, delta values (days to decrease virus concentration
by 1 log) ranged from 0.62–1.72 days, but there were no differences on virus survival among feed samples with or
without additives at the manufacturers recommended concentrations. Doubling the concentration of the additives
reduced the delta value to ≤ 0.28 days (P < 0.05) for all the additives except for Amasil (delta values of 0.86 vs. 4.95 days).
Feed additives that contained phosphoric acid, citric acid, or fumaric acid were the most effective in reducing virus
survival, although none of the additives completely inactivated the virus by 10- days post-inoculation.
Conclusions: Commercial feed additives (acidifiers and salt) may be utilized as a strategy to decrease risk of PDCoV in
feed, specially, commercial feed acidifiers at double the recommended concentrations reduced PDCoV survival in
complete feed during storage at room temperature. However, none of these additives completely inactivated the virus.
Keywords: Feed additives, Inactivation kinetics, Porcine delta coronavirus, Survival, Swine, Transmission, Virus
Background
There are three enteric coronaviruses that can cause
gastrointestinal illness in young pigs e.g., transmissible
gastroenteritis virus (TGEV), porcine epidemic diarrhea
virus (PEDV), and porcine delta coronavirus (PDCoV)
[1]. Transmissible gastroenteritis virus has been present
in the United States since 1946, but PEDV and PDCoV
were introduced more recently in 2013 and 2014, respectively. The spread of PEDV among swine herds was
* Correspondence: goyal001@umn.edu
2
Department of Veterinary Population Medicine, University of Minnesota, St.
Paul, MN 55108, USA
Full list of author information is available at the end of the article
rapid; and strict biosecurity measures known to prevent
transmission of other viruses such as porcine respiratory
and reproductive syndrome virus were ineffective; later
contaminated complete feed was demonstrated to be a
route for PEDV transmission that has been overlooked
in previous biosecurity protocols [2]. Therefore, for disease prevention purposes, it is essential to understand
proper feed handling procedures that minimize risk of
transmission, and to identify methods that can rapidly
inactivate these viruses if present in feed.
Commercial swine feed is often fortified with various
additives, including acidifiers such as organic and/or
inorganic acids to control bacterial and mold growth in
© The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
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reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
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(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Cottingim et al. Porcine Health Management (2017) 3:5
feed, increase growth performance of animals, improve
nutrient digestibility, and control harmful bacteria in the
animal gut [3]. Acidifiers are often added to feed as an
alternative to the use of antibiotics as growth promoters
and to control pathogens such as Salmonella spp. [4, 5].
Nursery pigs are believed to obtain the greatest benefit
from the addition of acidifiers, and the addition of acidifiers has been shown to increase growth rate by 12% [6].
Acidifiers are also effective in reducing diarrhea and
mortality while maintaining adequate growth of nursery
pigs [6]. This study was conducted to determine if the
addition of commercially available feed additives (salt,
sugar, and acidifiers), at recommended or double the
recommended concentrations, is effective in reducing
the survival of PDCoV in feed.
Methods
Virus propagation
The strain of PDCoV was obtained from the National
Veterinary Services Laboratory (NVSL; Ames, IA). Stock
virus was propagated in swine testicular cells. The cells
were grown in Minimum Essential Medium with Earle’s
salts supplemented with L-glutamine (Mediatech, Herndon,
VA), 8% fetal bovine serum (Hyclone, South Logan, UT),
50 μg/mL gentamicin (Mediatech), 150 μg/mL neomycin
sulfate (Sigma, St. Louis, MO), 1.5 μg/mL fungizone
(Sigma), and 455 μg/mL streptomycin (Sigma). The maintenance medium included 5 μg/mL of trypsin (Gibco, Life
technologies, Grand Island, NY) and the same antibiotics
as previously described. Cells inoculated with the virus
were incubated at 37 °C under 5% CO2 and were observed
for the appearance of virus-induced cytopathic effects
(CPE) for up to 6 days post-infection. The infected cells
were subjected to 3 freeze-thaw cycles (−80 °C/25 °C)
followed by centrifugation at 2500 × g for 15 min at 4 °C.
The supernatant was collected, aliquoted, and stored
at −80 °C until use.
Virus titration
Serial 10-fold dilutions of all samples were prepared in
maintenance medium followed by inoculation in monolayers of swine testicular cells contained in 96-well microtiter plates (Nunc, NY, USA) using 100 μL/well and 3
wells per dilution. Inoculated cells were incubated at 37 °C
under 5% CO2 for up to 6 days and examined daily under
an inverted microscope for the appearance of CPE. The
highest dilution showing CPE was considered the end
point. Virus titers were calculated as Tissue Culture
Infectious Dose TCID50/mL by the Karber method [7].
Feed matrix and laboratory analysis
The CGI Enhance ground commercial starter feed used in
this experiment was obtained from VitaPlus (Madison,
WI). This feed is designed for feeding pigs from 5–10 days
Page 2 of 7
post-weaning and does not contain any animal derived byproducts. The feed was confirmed to be negative for
PDCoV by real time reverse transcription-polymerase
chain reaction (RT-PCR). A sample of the feed was submitted to Minnesota Valley Testing Laboratories (New
Elm, MN), where dry matter (DM; method 930.15), ether
extract (method 2003.05), crude protein (CP; method
990.03), crude fiber (method 920.39), and ash (method
942.05) were analyzed following standard procedures [8].
The chemical analysis results of the feed were 91.43%
DM, 4.47% EE, 24.2% CP, 2.02% crude fiber, and 9.45%
ash on as is basis.
Feed additives
Six commercial feed acidifiers, UltraAcid P, (Nutriad,
Dendermonde, Belgium), Activate DA (Novus International,
St. Charles, MO), Acid Booster (Agri-Nutrition, DeForest,
WI), Kemgest (Kemin Agrifoods, Des Moines, IA), Luprosil
(BASF, Florham Park, NJ), and Amasil (BASF, Florham
Park, NJ) were evaluated when added at their manufacturers’ recommended concentrations (Table 1). In
addition, the effect of sodium chloride and sucrose on
virus survival was also evaluated. In a second experiment,
PDCoV survival was evaluated by adding the double of
the recommended amounts of these feed additives.
Virus inoculation procedure
Forty-eight aliquots of feed (5 g/aliquot) were placed in
plastic scintillation vials and the recommended concentrations of each feed additive were added. There were a total
of 8 observations at each of the 6-time point for each of
the 9 dietary combinations (control and 8 additives). Another set of 40 aliquots of feed were used at double of the
recommended concentrations of the additives, for a total
of 8 replications per each of the 5-time points and 9 dietary combinations (Table 1). Subsequently, 1 mL of PDCoV
(initial titer 3.2 × 105 TCID50/mL) was added to all vials.
The control treatment consisted of vials containing feed
and virus but no feed additive. The samples were thoroughly mixed using a vortex mixer and stored at room
temperature (~25 °C). An individual vial served as the experimental unit, and one vial from each set was removed
at 0, 7, 14, 21, 28, and 35 days to determine the degree of
virus inactivation. In the experiment involving double the
recommended concentrations of additives, samples were
removed and evaluated for virus inactivation at 0, 1, 3, 7,
and 10 days. Different time points were selected to account for greater virus inactivation in the early stages of
inoculation. To determine the amount of virus inactivation at each time point, the surviving virus in each vial
was eluted by adding 10 mL of 3% beef extract-0.05 M
glycine solution at pH 7.2. After thorough mixing by vortexing, the vials were centrifuged at 2500 × g for 15 min.
Serial 10-fold dilutions of the supernatants (eluates) were
Cottingim et al. Porcine Health Management (2017) 3:5
Page 3 of 7
Table 1 Commercial name of feed additives, active ingredients, concentration when mixed with complete feed at the manufacturers’
recommended doses (1×) and twice the manufacturers’ recommended doses (2×) along with pH of the diet and additive mixture
Feed additive (Manufacturer); (Active ingredients)
pH1
Amount
1×
2×
1×
2×
Complete feed
0
0
5.82c ± 0.02
5.82c ± 0.02
UltraAcid P (Nutriad, Dendermonde, Belgium);
(orthophosphoric, citric, fumaric, and malic acids)
150 mg
300 mg
5.84c ± 0.03
5.78c ± 0.02
Acid Booster (Agri-Nutrition, DeForest, WI);
(phosphoric, citric, and lactic acids)
10 mg
20 mg
5.84c ± 0.02
5.84cg ± 0.05
KEMGEST (Kemin Agrifoods, Des Moines, IA);
(phosphoric, fumaric, lactic, and citric acid)
10 mg
20 mg
4.20e ± 0.03
3.98e ±0.03
Activate DA (Novus International, St. Charles, MO);
(fumaric, benzoic, and 2-hydroxy-4-methylthiobutanoic acids)
20 mg
40 mg
5.50b ± 0.03
5.11b ± 0.02
Luprosil (Propionic acid, BASF, Florham Park, NJ);
(99.5% propionic acid)
56 μl
112 μl
5.74d ± 0.03
5.67d ± 0.03
Amasil (Formic Acid, BASF, Florham Park, NJ);
(61% formic acid, 20.5% sodium formate, 18.5% water)
46 μl
92 μl
5.88c ± 0.03
5.88gh ± 0.01
Sugar (Shoppers Value, Eden Prairie, MN); (sucrose)
20 mg
40 mg
3.22f ± 0.04
2.93f ± 0.02
Salt (Essential Every-day, Eden Prairie, MN); (sodium chloride)
20 mg
a
40 mg
4.93 ± 0.05
4.39a ± 0.03
1
Results shown are means of three replications; different superscripts differ at (P < 0.05)
inoculated in swine testicular cells as previously described
for virus titration. The amount of surviving virus was
calculated and compared with that in control vials (no
additive) and was expressed as log10 TCID50/mL. All treatments were applied and analyzed in triplicate.
Measurement of pH
Fifty mL of distilled water was added to 5 g of feed contained in a 100 mL glass flask. The feed suspension was
stirred at room temperature for 2 h using a magnetic
stirrer. The pH was measured using a pH probe (Fisher
Scientific, Waltham, MA) at 0, 15, 30, 60, and 120 min.
The final pH value was calculated as the average of the
values at different time intervals. The average pH for
feed was 5.82 ± 0.02 and this value was used to compare
the pH values after the addition of feed additives.
Mathematical models
Inactivation kinetics data (log TCID50/mL) were analyzed by using GInaFIT software, a freeware add-on for
Microsoft Excel (Microsoft, Redmond, WA) [9]. The
traditional log-linear model developed by Bigelow and
Esty (1920) was used to characterize the survival curves
of PDCoV by using the following equation [10]:
reduce initial virus titer by 90% or 1 log at a certain
temperature) and was calculated as:
D ¼
2:3
k
ð2Þ
The Weibull distribution function has been used to
describe non-linear inactivation patterns of different microorganisms after thermal and non-thermal processing.
Assuming that the temperature resistance of the virus is
governed by a Weibull distribution, Mafart et al. [11]
developed the following equation [12]:
Log ðN Þ ¼ logðN 0 Þ−
t n
ð3Þ
δ
where N is the surviving virus titer after treatment, N0 is
the initial virus titer, δ is the time (min or days) of first
logarithm decline in virus titer, and n is the shape parameter. The n value provides an indication of the shape
of the response curve. If n > 1, the curve is convex (it
forms a shoulder-shaped response), if n < 1, the curve is
concave (it forms a tail-shaped response), and if n = 1,
the curve is a straight line and can be described by a linear model.
Statistical analysis
Log N ¼ Log N 0 − ðk tÞ
ð1Þ
where N is the amount of surviving virus after treatment, N0 is the initial virus titer, k is the kinetic parameter (day−1), and t is the treatment time (d). The kinetic
parameter k is usually expressed as D, which is also
known as ‘decimal reduction time’ (time required to
Three replicates per treatment were used to determine
how well the model fit the experimental data by calculating the Adj. R2 defined as follows:
2
Adj: R2 ¼ 41 −
ðm−1Þ 1 −
SSQregression
SSQtotal
m − j
3
5
ð4Þ
Cottingim et al. Porcine Health Management (2017) 3:5
where m is the number of observations, j is the number
of model parameters, and SSQ is the sum of squares.
The effect of different additives on the kinetic parameters and survival of virus was assessed by using a mixed
model (SAS, v9.3; SAS Inst. Inc., Cary, NC) that included the effect of additives and time as fixed effects
and replicate/batch as random effects. Each vial was
considered as the experimental unit. Data were analyzed
for outliers and the presence of a normal distribution
using the UNIVARIATE procedure of SAS that calls for
calculations of sample moments, measurements of location and variability, standard deviation, test for normality, robust estimates on scale, missing values among
others. The LSMEANS statement in SAS was used to
calculate treatment means adjusted for model effects,
while Tukey’s test was used to determine differences
among treatments. For this study, significance was considered when P < 0.05.
Results
Effect of additives on the survival of PDCoV in feed at
their recommended concentrations
The goodness of model fit was analyzed by comparing the
Adj. R2 values from the log-linear and Weibull models.
The Adj. R2 values for the log-linear model (0.48–0.57)
were less than those obtained for the Weibull model
(0.86–0.93), indicating that the Weibull model provide a
better fit of the experimental data (Table 2). This is
explained mainly because the appearance of a resistant
fraction of the virus that was able to survive longer than
the length of the experiment (35 days). This residual survival produced long tails in the survival curves characterized by shape parameters (n) less than 1. This nonlinear
behavior resulted in D-values that overestimated virus survival (14.13–15.52 days), while the delta values obtained
with the Weibull model were between 0.86 and 1.72 days.
Weibull prediction values showed much faster inactivation
Page 4 of 7
kinetics and thus characterized better the virus survival
curves.
In spite differences in virus inactivation kinetics, none
of the additives appear to be effective in completely
inactivating the virus. The total amount of virus inactivation over the sampling period of 35 days was 3 log reduction for the control sample and all the additives
evaluated, indicating that none of the additives added at
the manufacturers’ recommend doses were effective in
reducing PDCoV survival.
Effect of additives on the survival of PDCoV in feed at
twice the recommended concentration
Doubling the concentrations of feed additives resulted in
faster PDCoV inactivation kinetics (0.0004–0.28 days) for
all additives, except for sucrose and formic acid (Table 3).
UltraAcid P and KEMGEST provided faster initial virus
inactivation kinetics than the other additives, and the delta
values were estimated to be 35 s. However, most of the
survival curves suggested that a large fraction of the virus
remained resistant to the treatment with the appearance
of tails (n values < 1) and a maximum inactivation degree
achieved of 2 log after 10 days of storage. The addition of
Luprosil (0.06 days), Acid Booster (0.28 days), and sodium
chloride (0.09 days) resulted in the greatest virus inactivation with 2.3-3.0 log reduction after 10 days of storage at
room temperature.
The pH of the complete feed without addition of acidifiers was greater than pH of the same complete feed with
the addition of Luprosil, Activate DA, KEMGEST, Acid
Booster, and Amasil. The pH of the complete feed with
addition of UltraAcid P was not different from that of the
complete feed. There was no correlation between the pH
values of the diet with the addition of acidifiers and the inactivation kinetics of PDCoV (delta values; Fig. 1). Interestingly, the virus appeared to survive better at pH values
lower than 3 and at pH 7 to 8.
Table 2 Kinetic parameters and correlation coefficients corresponding to the log-linear and Weibull models fitted to survival curves of
Porcine Delta coronavirus (PDCoV) in complete feed and feed additives included at the manufacturers’ recommended concentrations
Log-linear model
1
Weibull model
Additive
Log reduction (35 days)
D-value
Adj R
Delta (days)
Shape parameter (n)
Adj R2
Control
3.0
14.73 ± 1.04
0.55
0.86 ± 0.64
0.27
0.92
UltraAcid P
3.0
15.52 ± 2.09
0.48
0.62 ± 0.56
0.23
0.89
Acid Booster
3.0
14.41 ± 0.90
0.57
1.72 ± 1.85
0.32
0.86
KEMGEST
3.0
14.73 ± 1.04
0.55
0.86 ± 0.64
0.27
0.92
Activate DA
3.0
14.73 ± 1.04
0.55
0.86 ± 0.64
0.27
0.92
Luprosil
3.0
14.13 ± 0.90
0.55
1.00 ± 0.79
0.29
0.93
Formic Acid
3.0
14.73 ± 1.04
0.55
0.86 ± 0.64
0.27
0.92
Sugar
3.0
14.73 ± 1.04
0.55
0.86 ± 0.64
0.27
0.92
Salt
3.0
14.41 ± 0.90
0.57
1.70 ± 1.85
0.32
0.89
1
2
UltraAcid P, (Nutriad, Dendermonde, Belgium), Activate DA (Novus International, St. Charles, MO), Acid Booster (Agri-Nutrition, DeForest, WI), Kemgest (Kemin
Agrifoods, Des Moines, IA), Luprosil (BASF, Florham Park, NJ), and formic acid (BASF, Florham Park, NJ)
Cottingim et al. Porcine Health Management (2017) 3:5
Page 5 of 7
Table 3 Kinetic parameters and correlation coefficients corresponding to the Weibull model fitted to PDCoV survival curves in
complete feed and feed additives that were added at twice the manufacturers recommended concentrations
Log-linear model
1
Additive
Log reduction (10 days)
1
D-value
Weibull model
Adj R
2*
Delta2 (days)
be
Shape parameter (n)
Adj R2*
Control
2.0
6.05 ± 0.00
0.46
0.35 ± 0.00
0.23
0.86
UltraAcid P
2.0
7.42 ± 0.00
0.22
0.0004a ± 0.00
0.05
0.99
Acid Booster
2.7
4.65 ± 1.24
0.59
0.28be ± 0.18
0.27
0.93
KEMGEST
2.0
7.42 ± 0.00
0.22
0.0004a ± 0.00
0.05
0.99
Activate DA
2.0
6.74 ± 0.60
0.18
0.12bd ± 0.20
0.13
0.72
Luprosil
2.3
4.97 ± 2.40
0.27
0.06b ± 0.03
0.13
0.69
ac
Formic Acid
2.0
8.52 ± 0.00
0.08
4.95 ± 0.00
0.02
0.50
Sugar
2.0
10.00 ± 0.00
0.13
4.94ac ± 0.00
0.07
0.17
0.22
0.91
Salt
3.0
4.41 ± 0.52
0.55
bd
0.09
± 0.02
1
UltraAcid P, (Nutriad, Dendermonde, Belgium), Activate DA (Novus International, St. Charles, MO), Acid Booster (Agri-Nutrition, DeForest, WI), Kemgest (Kemin
Agrifoods, Des Moines, IA), Luprosil (BASF, Florham Park, NJ), and formic acid (BASF, Florham Park, NJ)
a, b, c, d
Means of 3 replications; different superscripts differ at (P < 0.05)
e
Trend comparing 2× Acid Booster vs. control (P < 0.1)
Discussion
Organic, inorganic, or blends of acids are commonly
added to swine feeds to control pathogens such as
Salmonella spp. [13]. To our knowledge, this is the first
study that has evaluated the impact of commercially available acids, sodium chloride, and sucrose on the survival of
PDCoV in swine feed. When these commercial additives
were added at the manufacturers’ recommended doses,
none of them were effective in decreasing survival of
PDCoV, we had to add all acidifiers at twice the manufacturer recommended concentrations to observe inactivation of PDCoV in complete swine feed. In contrast, PEDV
is inactivated by similar acidifiers at the manufacturers’
recommended concentration; Activate DA (0.81 d) and
KEMGEST (3.28 d) produced inactivation PEDV that was
faster than inactivation in the control diet [14].
The current experiment focused on determining inactivation kinetics of commercial additives available to the United
States feed industry, and did not focus on evaluating the
specific active ingredients present in these additives that
may inactivate PDCoV. However, based on the description
and order of the active ingredients listed for each
Fig. 1 Correlation of pH and delta value on virus inactivation at
double the recommended concentration of feed additives
commercial additive, it appears that some form of phosphoric acid (pKa 6.9 × 10 −3) was present in UltraAcid P
and KEMGEST, which suggests that this acid may be potentially responsible for inactivation of PDCoV. Phosphoric
acid has been shown to inactivate pathogens such as Salmonella spp. on stainless steel surfaces, but there are no
data available on inactivation of viruses in animal feed [15].
Inactivation of PDCoV was greater in the presence of
KEMGEST than Acid Booster, but the active ingredients in
these two feed additives are similar, with the exception of
fumaric acid present in KEMGEST. Furthermore, fumaric
acid was also present in UltraAcid P, which was also effective
in rapidly inactivating PDCoV. Therefore, it is possible that
fumaric acid in KEMGEST and UltraAcid P may be the primary component that causes PDCoV inactivation. Studies
have shown that fumaric acid is an effective antimicrobial
that reduces survivability of E. coli [16] and Salmonella spp.
[17]. It is believed that changes in pH affect viruses by increasing sensitivity to deoxyriobonuclease [18] and by altering the virus capsid by the loss of structural proteins [18].
The RNA of RNA-containing viruses (such as PDCoV) is
sensitive to ribonuclease at all pH levels tested (pH 3–9)
[19]. At pH levels of 5 and 7, RNA was hydrolyzed and there
was an absence of ribonuclease. There is no clear pattern or
indication of a specific acid that inactivates PDCoV and
more research is needed to depict the acid or combination
of acid that can completely inactivate the virus.
Comparing data from this experiment with data on inactivation of PEDV, it appears that PDCoV is more labile
than PEDV to environmental temperature and storage
conditions because the delta values for PDCoV were, in
general, much less (<2 d) than 17 days observed for PEDV
[20]. Comparison of inactivation kinetics suggest that
PEDV resists inactivation during feed storage to a greater
extent than does PDCoV. There are limited data
Cottingim et al. Porcine Health Management (2017) 3:5
comparing the survival of enteric coronaviruses in the environment, but after the initial outbreak of each virus,
PEDV infected more number of herds than PDCoV, this
epidemiology and geographic distribution data suggest
that PEDV survives longer than PDCoV and in agreement
with observations of the current experiment [21, 22].
Addition of salt, but not sugar, to the control diet caused
a decrease in delta values for inactivation of PDCoV. This
observation is in agreement with inactivation of PEDV in
complete swine feed, where adding both salt and sugar increased inactivation of PEDV [20]. Likewise, this observation is in agreement with results from an experiment that
suggest that addition of phosphate supplemented salt mix
to casting for sausage manufacturing increases inactivation
of several viruses affecting swine such as Food and Mouth
Disease Virus, Classical Swine Fever Virus, Swine Vesicular
Disease Virus, and African Swine Fever Virus [23].
Conclusions
Using feed acidifiers could be an effective strategy to
decrease the concentration of PDCoV in swine feed, but
double the manufacturer’s recommended concentration
was required to observe an effect. Using feed acidifiers
could be an effective strategy to decrease the concentration of PDCoV in swine feed, but double the manufacturer’s recommended concentration was required to
observe and effect. In spite the observed results on inactivation of PDCoV more experiments are needed to
demonstrate the effectiveness of these treatments as
means of preventing PDCoV transmission in feed on
more applied settings. None of the treatments applied in
this experiment were completely effective in inactivating
PDCoV. Therefore, the strategy proposed in this research should be used in combination with other virus
inactivation procedures within the processing and distribution steps for swine feed rather than a single kill step
for virus inactivation.
Abbreviations
Adj. R2: Adjusted coefficient of correlation; CP: Crude protein; DM: Dry
matter; EE: Ether extract; PDCoV: Porcine delta coronavirus; PEDV: Porcine
epidemic diarrhea virus; TCID: Tissue culture infectious dose
Acknowledgements
We thank Nhungoc Ti Luong for technical assistance in conducting this study.
Funding
We thank the National Pork Board for partial funding of this project and
Cenex Harvest States for the fellowship provided to K.M. Cottingim.
Availability of data and materials
Please contact author for data requests.
Authors’ contributions
KMC collected the data and wrote the manuscript, HV collected data and
revised the manuscript, PEU designed the experiments, analyzed data and
revised the manuscript, FS analyzed the data and revised the manuscript,
GCS revised the manuscript, SMG designed the experiments, collected data,
Page 6 of 7
analyzed data, and revised the manuscript. All authors read and approved
the manuscript.
Authors’ information
SMG, HV, FS College of Veterinary Medicine, PEU, GCS, and KMC College of
Food Agriculture, and Natural Resources Science at the University of
Minnesota.
Competing interests
The authors declare that they have no competing interest.
Consent for publication
Not applicable.
Ethics approval
Not applicable.
Author details
1
Department of Animal Science, University of Minnesota, St. Paul, MN 55108,
USA. 2Department of Veterinary Population Medicine, University of
Minnesota, St. Paul, MN 55108, USA.
Received: 10 September 2016 Accepted: 1 December 2016
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