Within- and between pen
transmission of Actinobacillus
Pleuropneumoniae in weaned
piglets.
Research project Veterinary Medicine University Utrecht
H.M.F. de Louw
3259471
26-8-2011
Project Tutor: Drs. T.J. Tobias
Within- and between pen transmission of Actinobacillus pleuropneumoniae in weaned piglets
Table of contents
Summary
page 3
Introduction
Clinical symptoms
Epidemiology
This survey
page
page
page
page
4
4
4
5
Materials and Methods
The farm
Bram’s research project
The animals
Sample collection
Laboratory analysis
Statistical analysis
page
page
page
page
page
page
page
6
6
6
6
7
7
8
Results
page 9
page 9
page 10
page 14
page 15
Status
The quantitative results of the PCR
Prevalence
Statistics
Discussion
page 17
Conclusion
page 20
References
page 21
Annex
1. Protocol DNA isolation
2. Protocol apxIVA qPCR
page 23
page 24
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Within- and between pen transmission of Actinobacillus pleuropneumoniae in weaned piglets
Summary
To investigate if the within pen transmission is more important than the between pen
transmission of Actinobacillus pleuropneumoniae, a longitudinal observational study on a
conventional farm was performed.
This research is part of a bigger project. The first part of the project, within- and between pen
transmission in the farrowing unit, was performed and results can be found in another report.
My research project focused on the transmission in weaned piglets. There were two batches of
piglets. We took tonsil swabs at the same age, approximately 3 weeks and 10 weeks of age.
The prevalence of PCR-positive pigs increased with age, the average prevalence of batch 1
and 2 together we found was at T = 4 33,6% and at T = 10 49%. Almost all pens are infected
at T = 10. Also we found an odds ratio for a status change from PCR-negative to PCR-positive
within a pen with positive pen mates of 8,7. All this points in the direction of within pen
transmission being more important than between pen transmission.
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Within- and between pen transmission of Actinobacillus pleuropneumoniae in weaned piglets
Introduction
Actinobacillus pleuropneumoniae is a gram-negative, hemolytic, fermentative and facultative
anaerobic coccobacillus of the Pasteurellaceae family1.
Contagious pleuropneumoniae, caused by Actinobacillus pleuropneumoniae, is a major
respiratory disease in pigs that causes severe economic losses to the pig rearing industry. The
economic losses are of great importance, mainly because of mortality, cost of treatment,
growth retardation and the higher prevalence of lung lesions and pleuritis in slaughter pigs2,3.
Until now little is known about the shedding and infection dynamics of Actinobacillus
Pleuropneumoniae in young piglets at farm level.
Clinical symptoms
The acute clinical symptoms of disease caused by Actinobacillus pleuropneumoniae include
open-mouth breathing with a blood-stained, frothy nasal, oral discharge, severe respiratory
distress, fever up to 41,5°C, reluctance to move, anorexia, typically leading to acute death
within 1-2 days. Pigs of all ages are affected and it causes worldwide high levels of mortality
4
. Findings at necropsy are a hemorrhagic necrotizing pneumonia and fibrinous pleuritis. In
chronically infected herds, the bacterium causes inefficient feed conversion, a decreased rate
of weight gain and by that an increased time to market5.
The severity of clinical disease depends on several factors. As mentioned above the disease
may be more prolonged and severe where more than one serotype is present and where
infection with other agents occurs. The virulence of the A. Pleuropneumoniae serotype, the
immune status of the pig and environmental factors play a role also.
Until now 15 different serotypes have been identified based on surface capsular
polysaccharide antigens. All serotypes contain several virulence factors such as Apx toxins,
capsules, transferrin binding proteins, adhesins, outer membrane proteins and secreted
proteases, which all play important roles in pathogenesis. Apx toxins are important virulence
factors because they are involved in the evasion of the host’s first line of defense and in the
formation of membrane pores in phagocytic and other target cells, resulting in osmotic
swelling and cell death 1.
Epidemiology
This disease occurs worldwide particularly in growing pigs from 2 to 6 months of age, even
though pigs of all ages are susceptible6. Seroepidemiological surveys have found that pigs in
70% of herds may have antibodies to one or more of several recognized serotypes of the
organism7.
The situation in the Netherlands
According to a pilot survey performed by the GD Deventer in 2005 the on farm prevalence of
Actinobacillus pleuropneumoniae serotype 2/9 in the Netherlands is 35,6% 8. According to
the study of Wellenberg et al, which contains the results of 500 sera of 100 randomly chosen
Dutch herds, 71,6% of the samples tested positive on one of the 15 serotypes of App9.
Transmission
There are two methods of transmission for A. pleuropneumoniae known, through direct
contact and by aerosols10. The organism spreads from animal to animal particularly during the
oral contact associated with fighting after mixing11.The organism survives in tonsils for 4-6
months or more and in lung lesions in pigs for weeks. In asymptomatic or sero-negative
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Within- and between pen transmission of Actinobacillus pleuropneumoniae in weaned piglets
carrier piglets or in the chronic form, apparently healthy pigs may harbor A.
pleuropneumoniae in their upper respiratory tract, particularly in their tonsils6, 7.
Aerosol Transmission
The study of Jobert et al. gives evidence of airborne transmission of A. pleuropneumoniae
over a distance of at least 2.5m. 2. Disease may be more prolonged and severe where more
than one serotype is present and where infection with other agents such as PRRS, PCV2 and
PRCV occurs. The organism survives for hours in aerosols, for long periods in clean water
(30 days at 4ºC) and appears to be rapidly killed by disinfectants in the absence of protein7.
Transmission between farms
Spread between farms occurs through the introduction of carrier animals to naive populations
12, 7
. There is no evidence for aerosol transmission between farms 11.
Transmission within farms
The prevalence of infection on conventional farms continues to increase, presumably due to
confinement rearing, crowding, inadequate ventilation, close contact and mixing of pigs. The
incidence of clinical disease is much lower than the prevalence of infection7.
Maes et al investigated the influence of herd factors on the within-herd seroprevalence of A.
pleuropneumoniae serovar 2,3 and 9. Serovars 2 and 9 are common in continental Europe,
while serovar 3 is common in England7. Maes et al. found several risk factors for the withinherd seroprevalence such as poor biosecurity measures (serovar 2 and 9) and purchasing gilts
from >1 origin herds (serovar 2). The seroprevalence of serovar 3 was significantly higher in
herds without a growing unit and in herds with a direct air-entry into the finishing unit 13.
This survey
Our survey took place at a conventional Dutch pig farm. This study focused on the withinand between pen transmission of Actinobacillus pleuropneumoniae in weaned piglets. If the
within pen transmission is more important this means that the main source of infection is in
the same pen as the animal which becomes infected. However if the between pen transmission
is more important it means that the main source of infection is not in the same pen as the
animal which becomes infected.
With this survey we hope also to find an indication if the animals are infected before the
clinical outbreak or become infected during the outbreak. If a farm has an outbreak of disease
caused by Actinobacillus pleuropneumoniae, there are quickly a lot of animals that become
sick. This indicates that if the animals become infected during the outbreak, there almost has
to be between pen transmission by aerosols. If it is known when the animals become infected,
there are ways to prevent infection.
So we hypothesize that within pen transmission is more important than between pen
transmission to cause a clinical outbreak of disease.
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Within- and between pen transmission of Actinobacillus pleuropneumoniae in weaned piglets
Materials and Methods
The farm
Samples are taken from approximately three and ten week old piglets of a conventional pig
rearing farm in the Netherlands. The farm consists of 1700 sows (Topigs 20), finishers and
breeding gilts. The sows are housed individually during gestation and lactation. This farm was
selected on history of respiratory disease and the farmer had to agree with our protocol.
During the trial we checked almost every week the piglets in our survey on clinical signs of
respiratory disease.
Bram’s Research Project
In part one of this trial the Actinobacillus pleuropneumoniae load was determined in tonsil
swabs from 38 and 26 sows by use of qPCR three weeks pre partum. All of the sows tested
positive. Two batches of 18 randomly chosen litters were followed during the lactation period.
Some studies have indicated that for many of the micro-organisms involved in respiratory
diseases vertical transmission, relying on sow to piglet contamination, is suspected to occur in
endemically infected herds during the suckling phase14. Because the aim of this study is to
determine the transmission within pens it was important that the composition of the pen
before and after weaning stayed the same. Three days before weaning a tonsil swab was taken
from the litters. Results with respect to sow to piglet transmission can be found in the report
of Bram Goesten.
The animals
Two batches were used to increase the power of the study and to check if the clinical trial is
repeatable.
Batch 1 consisted of 178 individually marked weaned
piglets in 16 pens. Nose-to-nose contact among pigs in
adjacent pens was possible (photo 1). Pen 1 to 12
contained piglets from one litter. Pen 13 to 16
contained piglets from different litters. The average
age at first sampling was 23,4 (±1,9) days. Three days
after the first tonsil swab was taken, the piglets were
weaned. At weaning the piglets were transferred litterwise to a growing unit. The second tonsil swab was
taken from 176 piglets at an average age of 65,1 (± 3,6)
days.
Photo 1: housing of batch 1
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Within- and between pen transmission of Actinobacillus pleuropneumoniae in weaned piglets
Batch 2 consisted of 142 individually marked
weaned piglets in 12 pens. Nose-to-nose contact
among pigs in adjacent pens was limited to the
minimum (photo 2). All pens contained piglets from one
litter. The average age at sampling was 21,6 (± 3,6)
days. Three days after the first tonsil swab was taken,
the piglets were weaned. At weaning the piglets were
transferred litter-wise to a growing unit. The second
tonsil swab was taken from 139 piglets at an average
age of 63, 6 (± 2,9) days. It was necessary to take a third
tonsil swab of batch two. This swab was taken from
138 piglets at an average age of 73, 6 (± 2,9) days.
Photo 2: housing of batch 2
Sample collection
During sample collection hand gloves and boot covers were replaced between different pens
in order to minimize the risk of transmission between pens during sample collection.
Tonsil swabs:
At the age of 4 and 10 weeks a tonsil swab was taken from each piglet. At 4 weeks of age the
animals were restrained by a person, at 10 weeks by placing a conventional cable snare over
the maxilla. The surface of the tonsils was swabbed for 10 seconds with a sterile toothbrush.
All samples were placed in sterile 35 mL tubes. They were individually identified and placed
overnight in 4ºC. The conventional cable snare was placed in alcohol between the sampling of
two animals. A negative control sample of the alcohol was taken at the end of sampling.
Laboratory analysis:
Protocol processing toothbrushes.
10 mL of saline was added to all samples. They were mixed for 15 minutes and 1 mL for
DNA isolation and 1 mL for storage was put into an eppendorf cup. These were individually
marked and stored overnight at -20 ºC. A negative control of saline was included.
Protocol DNA isolation.
After thawing the samples were centrifuged for 5 minutes. The supernatant was discarded and
200µL Instagene Matrix was added. After short mixing, the samples were heated for 30
minutes at 56 ºC. After mixing for 10 seconds the samples were heated for 8 minutes at 100
ºC. After another 10 seconds mixing the samples were centrifuged for 5 minutes at 13000g
and stored at -20 ºC. The complete protocol is attached in annex one.
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Within- and between pen transmission of Actinobacillus pleuropneumoniae in weaned piglets
Protocol apxIVa qPCR.
Before qPCR analysis, samples were thawed, briefly mixed and centrifuged for 5 minutes at
13000g. Mix was made of TaKaRaMix, forward and reverse primers (APXIVANEST1-F and
APXIVANEST1-R15), probe and milliQ. The PCR plate contained 15µL mix and 10µL
sample. All samples were tested in duplicate. The plate was covered with a film and
centrifuged for 10 seconds at 500g. For qPCR analysis a BioRad iQ5 thermocycler (BioRad
Laboratories B.V.) was used. The program of the PCR began with an initial denaturation (60
seconds at 95°C), followed by 40 cycles of 10 seconds at 95°C, 30 seconds at 56°C and final
one minute at 40°C. The complete protocol is attached and can be found in annex two.
At each sampling, an animal was considered positive when the sample tested positive by
qPCR (sqmean ≥ 5). The quantitative result of the PCR is called Sq. All samples were tested
in duplo. In all analyzes the mean value of the Sq of both duplicates was used. Also the
logarithm of the mean value of Sq has been used. By taking the logarithm the data got divided
normally. The averages by pen were calculated with the logarithm of the mean value of Sq +1.
By using the logarithm of the mean value of Sq +1 also the negative animals were counted.
Statistical analysis
The prevalence was calculated on batch and pen level and was compared by prevalences
mentioned in literature. Logistic regression methods were used to analyze possible risks for a
piglet to become positive.
First we tested if a negative piglets has a higher odds for becoming positive if the piglet had
one or more positive pen mates at T = 4. Second was tested if the number of positive animals
mattered. Finally was tested if the number of positive pigs in adjacent pens mattered for
becoming positive.
The logistic regression was performed by SPSS 16.0. The output from SPSS gives EXP(B),
this is the odds ratio. Also in the output is shown ‘sig.’, which gives the significance of the
model. Finally we used the ‘CI’, which means 95% confidence interval of the odds ratio.
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Within- and between pen transmission of Actinobacillus pleuropneumoniae in weaned piglets
Results
Batch 2 has been sampled three times in stead of two like batch 1. In these results are written
the results of the first and third sample moment.
Status
The status of the animals after the first (t = 4) and second (t = 10/11) sampling is shown in
table 1 for batch 1 and in table 2 for batch 2.
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Within- and between pen transmission of Actinobacillus pleuropneumoniae in weaned piglets
The quantitative results of the PCR
Scatter outline of batch 1 and 2.
Batch 1:
At T = 4 28,6% of the piglets is
PCR-positive.
At T = 10 55,1% of the piglets is
PCR-positive.
Four pigs tested positive at T = 4
and negative at T = 10.
Batch 2:
At T = 4 38,7% of the piglets is
PCR-positive.
At T = 11 42,8 % is PCRpositive.
Thirteen pigs tested positive at
T = 4 and negative at T = 10.
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Within- and between pen transmission of Actinobacillus pleuropneumoniae in weaned piglets
Statistics
Result of the independent samples T-test on the quantitative PCR result of all positive animals
of batch 1 and 2 after first sampling moment (SPSS output 1a).
The difference in mean quantity of A. pleuropneumoniae DNA after first sampling is found
significant (p = 0, 036).
Result of the independent samples T-test on the quantitative PCR result of all positive animals
of batch 1 and 2 after second sampling moment (SPSS output 1b).
(2 PCR-positive piglets of batch 2 died between both sample moments)
The difference in mean quantity of A. pleuropneumoniae at second sampling is found
significant (p < 0, 01)
If we compare the outcome of the logarithm of Sqmean of batch 1 and 2 we see that batch 2
has an average significantly higher than batch 1 after first sampling. After second sampling
batch 1 has an average that is significantly higher than the average of batch 2.
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Within- and between pen transmission of Actinobacillus pleuropneumoniae in weaned piglets
In graph 3 and 4 is shown the average outcome of the PCR by pen. Graph 3 shows the result
of batch 1, Graph 4 the result of batch 2.
In graph 3 almost all averages increased. The average of pen number 8 decreased, but all
animals were positive at T = 4 and stayed positive. Pen 12 and 13 stayed negative during this
study.
In graph 4 the average of pen 1, 2, 3, 5, 8, 9, and 11 increased. Pen number 6 stayed negative
during this study. Pen numbers 4, 7, 10 and 12 decreased in average value. In pen 4 eleven
animals were positive at T = 4 and 12 at T = 10 only the average decreased. In pen numberr
10 the prevalence stayed the same, only the average value decreased. In pen 7 changed the
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Within- and between pen transmission of Actinobacillus pleuropneumoniae in weaned piglets
status of 3 animals and in pen 12 changed the status of 10 animals from PCR-positive to PCRnegative.
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Within- and between pen transmission of Actinobacillus pleuropneumoniae in weaned piglets
Prevalence
The prevalence per pen for each batch is shown in graph 1 and 2.
Graph 1 shows an increase in prevalence in all pens except pen 12 and 13, which stayed
completely negative during the trial. The average prevalence of batch 1 at T = 4 was 28,6%
and at T = 10 it was 55,1%.
Graph 2 shows generally the same as graph 1. In most pens the prevalence has increased.
Except pen 6, this one stayed negative during the trial. In pen 7 and 12 the prevalence
decreased. The average prevalence of batch 2 at T = 4 was 38,7% , and at T = 11 it was 42,8
%
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Within- and between pen transmission of Actinobacillus pleuropneumoniae in weaned piglets
Logistic regression analysis
The odds ratio for a status change from PCR-negative to PCR-positive within a pen with
positive pen mates was 8,7 (p< 0,001; CI: 4,2 - 17,9)(SPSS output 1). This indicates that a negative
piglet in a pen with one or more positive pen mates has a 8,7 times higher odds for becoming
positive than a negative piglet in a pen without positive pen mates.
To determine if the number of positive pen mates matter, another logistic regression was done
(SPSS output 2).
In the output above there isn’t anything significant. The category of zero positive animals in
the pen at T = 4 contained 103 animals. All other categories contain not enough animals to put
into a logistic regression analysis. That’s why another logistic regression was done(SPSS output 3).
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Within- and between pen transmission of Actinobacillus pleuropneumoniae in weaned piglets
In SPSS output 3 a “backward stepwise: likelihood ratio” method was used. This method
searches for the model which fits the best at the data entered. The model at step 2 fits the best
at the data. The odds for becoming positive is 5 times higher if there are two positive pen
mates at T = 4 instead of more or less than two positive pen mates. If there are more than two
positive pen mates the odds for becoming positive is 2,6 times higher than if there are less
than two positive pen mates. If there are zero positive pen mates at T = 4, it protects against
becoming positive, because the odds < 1.
To determine if the number of positive animals in adjacent pens matters, another logistic
regression was done (SPSS output 4,5).
In the output above also a “backward stepwise: likelihood ratio” method was used. The
number of positive animals in adjacent pens seems not to be important, because none of the
tested numbers gives a significant outcome (SPSS output 4). However if the number of piglets in
adjacent pens is combined with the presence of one or more positive pigs in the pen the output
changes (SPSS output 5).
If a negative piglet has > three positive neighbors and one or more positive pen mates the
odds for becoming positive is 4,3 times higher than when the piglet has 0,1,2 or 3 positive
neighbors and no positive pen mates (SPSS output 5).
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Within- and between pen transmission of Actinobacillus pleuropneumoniae in weaned piglets
Discussion
The aim of the present study was to describe the within- and between pen transmission in two
batches of weaned piglets. Two batches were tested to increase the power of the study. Tonsil
swabs were used because the use of the qPCR and especially because serum samples don’t
give information about the transmission of Actinobacillus pleuropneumoniae because of the
asymptomatic carrier pigs and the sero-negative carrier pigs.
This longitudinal observational study gives information about the prevalence of A.
pleuropneumoniae on a conventional Dutch pig farm at four and ten weeks of age. By no
mingling of piglets we tried to determine which transmission route is more important, withinor between pen transmission. If it is known when and in which manner the animals become
infected, there are ways to prevent infection. This study is important because nobody knows
when and how the piglets become infected and at the moment of a clinical outbreak it is too
late to intervene.
In this longitudinal observational study a control group was not included, because there were
not enough people available to help with sampling and processing in the lab. Also because of
the aim, to describe the within- and between pen transmission, there wasn’t a control group. A
control group would be a batch which was treated as usual in the farrowing unit, meaning that
the piglets were mixed and mingling of piglets can increase the between pen transmission7
The farm
The herd included in this study was not selected at random and may not be representative of
the population on Dutch conventional pig farms. It was selected because of the history of
respiratory disease. We performed this survey on a conventional farm instead of in
experimental setup. In this manner we could examine more pigs. Also an experimental setup
can never imitate the transmission of A. pleuropneumoniae at farm level.
The animals
The longitudinal observational study started with two farrowing units of 18 sows each. Before
weaning 12 litters per batch were selected to remain in the clinical trial. Selection of sows was
made based on parity. We didn’t want to continue the survey with, for example, litters from
young sows only. Also litters which were mixed during lactation period or which were
weaned earlier were discarded. From the remaining litters, 12 were selected random to remain
in the survey. We selected 12 litters because of the size of the unit after weaning. This unit
should contain 12 pens with minimal possibility for nose-to-nose contact between pens by
agreement.
However, at weaning of batch 1 the farmer had only place for the piglets in a unit which had
16 pens with the possibility for nose-to-nose contact. That’s why batch 1 consists of 12
complete litters and 4 mixed litters in one unit.
The twelve complete litters of batch 1 contained at T = 4 37% positive animals, at T = 10 this
has increased to 53%. The four mixed litters contained at T = 4 13% positive animals, at T =
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Within- and between pen transmission of Actinobacillus pleuropneumoniae in weaned piglets
10 this has increased to 45%. The increase is 16,5% by the complete litters versus 32% by the
mixed litters. This indicates that there has been more transmission by the mixed litters
probably from animal to animal during the oral contact associated with fighting after mixing.
Batch 2 has been sampled three times in stead of two like batch 1. In the results are written the
results of the first and third sample moment. The results of the second sample moment were
not plausible according to us. There were 23 animals that changed from positive at t = 4 to
negative at t = 10. So we tested the animals a third time, ten days after t = 10. In the results the
third sample moment is called t = 11.
Results
Quantitative PCR results
If we compare the outcome of the logarithm of Sqmean of batch 1 and 2 we see that batch 2
has an average significantly higher than batch 1 after first sampling. After second sampling
batch 1 has an average that is significantly higher than the average of batch 2.
The averages of the logarithm of the quantitative outcome of the PCR + 1 by pen are shown in
graph 3 and 4. In graph 3 is shown that pen number 8 decreases a little in average value. All
animals in pen 8 were positive at T = 4 and stayed positive, only the average decreased.
Actinobacillus pleuropneumoniae survives deep in the crypts of the tonsils17. So the pathogen
load on our swabs can vary, also a mistake by taking or processing the samples could be the
reason.
Because the average of batch 2 decreased between the sample moments, we believe it is
plausible that in graph 4 pen numbers 4, 7, 10 and 12 decreased in average value. In pen 7 and
12 together changed the status of 13 animals from PCR-positive to PCR-negative. In pen 4 1
animal became positive and the average decreased, it could be that the negative sample at T =
4 was false negative. In pen 10 all animals were positive at T = 4 and stayed positive, the
pathogen load on the tonsils can be changed17 or a mistake by taking or processing the
samples could be the reason of the decrease in average value.
Prevalence
In literature prevalences from slaughter pigs in the Netherlands ranging from 35 to 72% were
found8, 9. The prevalence found in this study over two batches was at approximately three
weeks of age 33,6%, and at approximately 10 weeks of age the prevalence increased to 49%.
The prevalence in batch 1, pen 12 and 13 excluded, and batch 2, without pen 6, 7 and 12,
increased like expected. The pattern of tonsillar colonization found is probably due to that few
pigs get infected by A. pleuropneumoniae during the nursering period. These infected pigs
spread the bacteria to other pigs after weaning when colostral antibodies against Actinobcillus
pleuropneumoniae decline.16. Pen 12 and 13 from batch 1 and pen 6 from batch 2 stayed
negative during the trial.
In pen 7 and 12 of batch 2 the prevalence decreased because the status of 3 piglets in pen 7
and 10 in pen 12 changed from PCR-positive to PCR-negative.
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Within- and between pen transmission of Actinobacillus pleuropneumoniae in weaned piglets
A status change from PCR-positive to PCR-negative has never been described in literature7.
Pigs that survive clinical illness become carriers, subclinical ill pigs also become carriers7, 6.
There are a few possible explanations how piglets in our trial became negative. It could be due
to incorrect sampling, incorrect processing in the lab or the samples may have been false
positive at first sampling. The piglets of batch 2 had been fed just before our sampling at T =
10. Maybe the liquid feed interferes with the qPCR.
After inquiries we find out that batch 2 had been fed liquid feed containing antibiotics. The
whole batch got ampicillin trihydrate three days at the end of week 6 of our study and another
two days at the end of week 7. Also they got doxycyclin three days at the beginning of week 7
of our study and another two days at the beginning of week 8. Batch 1 wasn’t been fed
antibiotics and there were only four animals that changed from PCR-positive to PCRnegative. Three piglets of the four had a qPCR result at first sampling near to five, which is
the cutoff value. So the results of these four pigs may have been false positive.
Batch 1 showed an increase in prevalence of 26,5% between the first and second sample
moment. On the other hand batch 2 showed an increase of only 4,1% between the first and
third sample moment. At T = 4 batch 2 showed already a prevalence of 38,7 % in contrast to
batch 1 which had a prevalence of 28,6% at T = 4. So the transmission in the farrowing unit
was higher in batch 2 and the transmission after weaning was higher in batch 1.
Logistic regression analysis
The logistic regression analysis in this report included all data of batch 1 and 2. The batches
were analyzed separately also. Batch 1 and 2 had different outcomes when they were analyzed
separately. These outcomes are not mentioned in this report because they don’t influence the
overall conclusions. We found a difference between the batches, but to know what caused this
difference further research is needed.
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Within- and between pen transmission of Actinobacillus pleuropneumoniae in weaned piglets
Conclusion
We hypothetisized that the within pen transmission is more important than the between pen
transmission at the moment of a clinical outbreak. In this study we found that the prevalence
at 10/11 weeks of age is already quite high in both batches (55, 1% and 42, 8%) and in both
batches almost all pens are infected at 10/11 weeks. Combined with the odds ratio’s for status
change in a pen with positive pen mates this points in the direction of within pen transmission
as more important than between pen transmission. Unfortunately we didn’t see a clinical
outbreak during our trial. At the moment of a clinical outbreak it still can be that between pen
transmission plays an important role.
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6. Savoye, C. et al. A PCR assay used to study aerosol transmission of Actinobacillus
pleuropneumoniae from samples of live pigs under experimental conditions. Vet. Microbiol. 73, 337347 (2000).
7. Gottschalk, M. & Taylor, D. J. Actinobacillus pleuropneumoniae in: Diseases of Swine (eds Straw,
E. J., Zimmerman, J. J., Allaire, S. D. & Taylor, D. J.) (Blackwell publishing, Iowa, USA, 2006) p.
563-576.
8. Dr. Geudeke, T., ir. van de Ven, S.C.G, dr. de Jong, M. & drs. Bouwkamp, F. Verhoging van
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met gebruik van systematisch serologisch onderzoek. (2005).
9. Wellenberg, G. J. et al. Profiles of Actinobacillus pleuropneumoniae serotypes in Dutch slaughter
pigs. (Proceedings of the 21st international pig veterinary society congress.)
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pleuropneumoniae infection in hunted wild boars (sus scrofa) in germany. J. Wildl. Dis. 46, 551-555
(2010).
11. Taylor, D. J. Pleuropneumoniae (Actinobacillus pleuropneumoniae infection) in Pig diseases 8th
edition (Glasgow) Great Britain, (2006) p. 207- 213.
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15. Schaller, A. et al. Identification and detection of Actinobacillus pleuropneumoniae by PCR based
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Within- and between pen transmission of Actinobacillus pleuropneumoniae in weaned piglets
16. Vigre, H., Angen, Ø., Barfod, K., Lavritsen, D. T. & Sørensen, V. Transmission of Actinobacillus
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83, 147-159 (2001).
Research project University of Utrecht: H.M F. de Louw
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Within- and between pen transmission of Actinobacillus pleuropneumoniae in weaned piglets
Annex 1: Protocol DNA isolation
Vooraf:
Monsters zo koud mogelijk (niet bevroren) mee naar lab nemen.
OPMERKINGEN:
NEGATIEVE controles meenemen
Van: fysiol. Zout, van Instagene Matrix
Gebruik handschoenen
Werk zorgvuldig en netjes
Elke keer:
NIEUW flesje fysiol.zout nemen
DNA isolatie met Instagene Matrix (Biorad)



Neem 200µl uit het epje met monstermateriaal en doe in nieuw epje
Sticker beide epjes; bignummer, datum, dag proef, soort monster, DNA of restant,
Neem 200ul fysiol. Zout als – procescontrole mee in een leeg epje



Centrifugeer 5 minuut bij 13,000 g
Pipetteer supernatant af met 200µl pipet.
Voeg 200µl Instagene Matrix toe (met 1 ml Pipet) per monster nieuwe pipetpunt) en vortex
kort
Neem 200µl Instagene mee als – procescontrole in nieuw epje.






Zet 30 minuten bij 56°C in hitteblok. (Voorverwarm ander hitteblok 100°C). Denk aan
temperatuur instelling en werkelijke temperatuur!
Na 30 minuten epjes 10 sec vortexen en
8 min in 100°C hitteblok zetten,
epjes vortexen en
Afdraaien: 5 minuten x 13.000g

Na afdraaien bewaren bij -20°C (NIET overpipetteren supernatant).
Bij ieder gebruik voor PCR wederom mixen en 5 min bij 13.000g afdraaien
DNA isolatie en qPCR mag NIET op dezelfde dag plaatsvinden
Per isolatie dus mogelijk 22 monsters + 1 fysiol.zout + 1 Instagene leeg.
Evt ook positieve controle!!
Research project University of Utrecht: H.M F. de Louw
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Within- and between pen transmission of Actinobacillus pleuropneumoniae in weaned piglets
Annex 2: Protocol apxIVA qPCR
Vooraf: alle benodigde DNA templates ontdooien, centrifugeren (13,000g x 5 min). En andere
benodigde materialen ontdooien.
OPMERKINGEN:
NEGATIEVE controles meenemen
Van: fysiol. Zout, van Instagene Matrix
Gebruik handschoenen
Werk zorgvuldig en netjes
Benodigdheden:
TaKaRa mix, primer (F en R), Probe, MilliQ
Koelblok uit -20°C, qPCR plaat + 1 sealfilm, roller
Pipetpuntjes voor PCR, pipetten (1000µl, 100µl en 10µl)
Protocol qPCR mix maken


In PCR ruimte 1; mix in 1 epje mengen en vervolgens in qPCR plaat pipetteren.
Mix betaat uit:
o TaKaRa mix
12,5 µl/sample
o Forward primer
0,5 µl/sample
o Reverse primer 0,5 µl/sample
o Probe
1,0 µl/sample
o MilliQ water
0,5 µl/sample
Prepareer 10% meer mix dan het aantal te bepalen monsters.
Vul de qPCR plaat met 15,0 µl / well.





In PCR ruimte 2; of flowkast
o Zet plaat met mix in ‘koel’blok.
o Open epjes op afstand, voorkom aerosol vorming.
o Voeg 10µl DNA template toe. Gebruik monsters in tweevoud.
o
Plak PCR film op de plaat en rol stevig aan.
Centrifugeer plaat 500g x 10 sec. KORT.
Zet plaat in qPCR apparaat (IQ5).
Zet in flowkast UV lamp aan voor min. 10 min.



Selecteer PCR programma: “dec. 2009”
Vul het ‘plaatschema’. En ‘Run’ PCR.
Na afloop plaat verwijderen en even in koelkast bewaren.

Gebruik ‘threshold’-value=200 voor analyse. Exporteer data naar Excel op USB stick.
Research project University of Utrecht: H.M F. de Louw
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