PREVALENCE OF AVIAN INFLUENZA AND IDENTIFICATION OF
RISK FACTORS IN POULTRY COLLECTOR FACILITIES IN
CIPUNAGARA, INDONESIA
A multi intervention pilot study, November 2010 – January 2011
Sandy van Kruysdijk
Student, faculty of veterinary medicine, University of Utrecht, the Netherlands
ACKNOWLEDGEMENTS
First I would like to thank Arief Ervana and Ni Made Ferawati for helping us communicate with
the Indonesian people, for keeping us save while driving us around in the busy and mad
Indonesian traffic, for assisting in our research project and for helping us to survive our two
month stay in Jati where our base camp was located.
I would also like to thank Unita Pratiwi, Adrianus Fardin Arut, Haryandhi Eka Putra and field
coordinator Kanda Muhamad. Thank you all for the great time we had.
Thanks to all the people at Bogor University, especially Ibu Ethi, Yusuf Ridwan, Chaerul Basri and
Denny W. Lukman for their advice, hospitality and for the great excursions they organised.
Also, I would like to thank the DINAS team for their support in organizing the project and for
assisting during the sample taking.
People from West Java Dinas Peternakan Laboratory in Cikole, especially Yoni and Afrida, thank
you for processing all our samples.
Off course I would like to thank the owners of the slaughterhouses for participating in this
project.
Special thanks to Dr. Patrick Hermans and Dr. Ivo Claassen for supervising the project, the
excursions, for letting us use a real shower and for the survival package you gave us in Jati. We
couldn’t have done this without your support.
Thank you Dr. Annamarie Bouma, without you we would not have been able to do our research in
Indonesia in the first place.
And last but not least I would like to thank Wytze Brandsma, my research partner, for his advice,
cooperation and the great time we had.
2
CONTENTS
Acknowledgements
Contents
Abstract
Introduction
Indonesian-Dutch Partnership program
Avian Influenza
Etiology
Transmission
Aim of this project
Materials and methods
Materials
Methods
Social visits
The sample taking and data gathering
Testing the field samples in the lab
Questionnaire analysis
Results
Questionnaire analysis
General information concerning the slaughterhouses
Specific information per slaughterhouse
Laboratory results
Discussion
Conclusion and recommendations
References
Appendix
3
ABSTRACT
Avian influenza is an endemic disease in Indonesia and outbreaks of highly pathogenic avian
influenza resulted in high mortality rates in poultry flocks and economic losses.
Due to the outbreaks in 2005 a partnership program was established between the Ministry of
Agriculture in Indonesia and the Dutch Ministry of Agriculture, Nature and Food Quality (LNV)
to assist the Indonesian government in the control of HPAI in Indonesia. The aim is to assist local
veterinarians and farmers to improve control measures and to get insight in the epidemiological
characteristics of the HPAI infection which might also contribute to improving existing control
strategies. The greatest threat in spreading the disease is mechanical transmission, especially due
to human involvement by transferring infective organic material from infected to susceptible
birds by movement of personnel, equipment and vehicles between farms.
The aim of this multi-intervention project was to investigate the incidence of HPAI and to
examine the most important causes of morbidity and mortality among poultry flocks located in
this district. In addition, risk factors which may contribute to the occurrence of disease were
identified.
Although no HPAI was found in this research area it is still very important to improve hygiene
and other preventive measures in order to avoid possible future spread of disease.
4
INTRODUCTION
Indonesian-Dutch Partnership program
Outbreaks of Highly Pathogenic Avian Influenza (HPAI) H5N1 virus among poultry flocks have
been reported since 1997. Since then the infection has spread to many countries in the world.
HPAI outbreaks are usually successfully controlled by stamping out infected flocks and several
other measures like a standstill and hygienic measures. However in some countries in Asia and
Africa the disease has become endemic (OIE, 2010). The disease can be devastating for the
poultry industry and public health officers also fear the disease for the pandemic threat of disease
in humans.
One of the countries where AI is endemic is Indonesia. Since the first outbreak was detected in
August 2003 the virus has spread rapidly across the country so fast that at present 31 out of 33
provinces are considered to be endemically infected. Outbreaks resulted in high mortality rates in
poultry flocks, and economic losses. The disease is now controlled by large scale vaccination
programs implemented for commercial poultry flocks. Small scale chicken holders however, do
not apply control measures. Despite control measures outbreaks are still reported, although the
number is limited and are only reported to occur in backyard flocks. In addition to the infection in
poultry, several humans became infected, of which more than 100 died from the infection (WHO,
2010).
In 2005, a partnership program was established between the Ministry of Agriculture in Indonesia
and the Dutch Ministry of Agriculture, Nature and Food Quality (LNV) to assist the Indonesian
government in the control of HPAI in Indonesia. As part of the activities of this IndonesianDutch partnership, a multi-intervention pilot project is to be conducted. This pilot project aims to
control HPAI outbreaks in one defined area and in all sectors of poultry production such as
commercial breeding farms, commercial broiler farms, slaughter and poultry collecting facilities,
nomadic duck flocks, and village backyard poultry flocks, using a variety of control measures.
The aim is to assist local veterinarians and farmers to improve control measures and to get insight
in the epidemiological characteristics of the HPAI infection which might also contribute to
improving existing control strategies.
The project took place in Cipunagara sub-district, Subang, West Java. Birds on commercial farms
are routinely vaccinated against HPAI, whereas birds in the surrounding villages are not. Many of
the commercial breeding farms in Subang District are located close to villages with poultry.
Frequent occurrences of sudden death events among this village poultry have been reported,
suggesting the presence of HPAI. Although it is unknown whether outbreaks of HPAI and/or ND
occur on commercial flocks, as owners do not provide the essential information, it seems to be
‘clear’ that outbreaks do occur in village poultry. Improvement of the poultry health and
associated income of local villagers is an important part of the Indonesia-Dutch partnership.
Avian Influenza
Etiology
Avian influenza are placed in the family of Orthomyxoviridae which have segmented, negativesense, single-stranded RNA genomes.1,2 The Orthomyxoviridae family consists of five genera:
Influenzavirus A, Influenzavirus B, Influenzavirus C, Thogotovirus and Isavirus.1
5
Only viruses of the Influenzavirus A genus are known to infect birds.1,2 Type A influenza viruses
are further divided into subtypes based on the antigenic relationships of the surface glycoproteins,
haemagglutinin (HA) and neuraminidase (NA). Sixteen HA subtypes (H1–H16) and nine NA
subtypes (N1–N9) have been identified. Most strains are of low pathogenicity and produce either
subclinical infections or respiratory and/or reproductive diseases in a variety of domestic and
wild bird species.2 Viruses of the H5 and H7 subtypes have been shown to cause HPAI which
results in high morbidity and mortality.1 It appears that most of these HPAI viruses originated
through mutation of the haemagglutinin surface protein from LPAI H5 and H7 viruses after they
were introduced into poultry from the wild bird reservoir.1,2 The factors that bring about mutation
from LPAI to HPAI are not known.1 It is impossible to predict if and when this mutation will
occur because in some instances, mutation seems to have taken place rapidly after introduction
from wild birds where in other occasions the LPAI virus progenitor circulated in poultry for
months before mutating. However, it can be reasonably assumed that the wider the circulation of
LPAI in poultry, the higher the chance that there will be a mutation to HPAI.1,3,4,5,6,7
Transmission
It seems that transmission from bird to bird occurs as a result of close proximity between infected
and naïve hosts. Direct contact with infected birds or with contaminated exudates or droppings
are necessary for infection to be transmitted from one bird to another. This also indicates that
airborne spread over large distances is an unlikely event.1 There is some evidence that airborne
spread during an outbreak in Pennsylvania 1983-1984 played a role amongst very closely
situated farms and that flying insects could become contaminated with infected feces and thus
spreading the AI virus mechanically.8,9,10,11,12,13
It is known that the probability of incursion of virus into a population depends on the frequency
of contact between birds. After introduction, secondary spread of the virus within the area is
usually associated with human involvement, probably by transferring infective organic material
from infected to susceptible birds by movement of personnel, equipment and vehicles between
farms.1,5,6,8,9,10,11,12,13,14,15,16
Clinical signs in poultry
Influenza infections in poultry can be asymptomatic, but it often causes production losses and a
range of clinical disease from mild to severe in affected flocks.
LPAI does not cause high mortality (2-3%) in affected flocks and can easily be confused with
other conditions. LPAI infections can go unnoticed but typically it causes mucosal infection in
the respiratory and/or enteric tract.1,5 Among the clinical signs rales, snicking and light coughing
may be observed.1 Sexually mature broiler breeders may have a fevered condition accompanied
by depression, somnolence and loss of appetite. This can be followed by a drop in egg
production. Cyanosis of the combs and wattles may be seen in a few birds in this stage. In an
acute stage of illness misshapen eggs and/or discoloring may be seen.
6
HPAI on the other hand causes severe
systemic infections which result in high
mortality in affected flocks.5 Flock mortality
may be as high as 100% within three to four
days from the onset of the first clinical
signs. Symptoms start with anorexia,
depression and a major drop in egg
production. These symptoms are followed
by nervous signs, prostration, reluctance to
move, tremors of the head, paralysis of the
wings and incoordination of leg movement.
Typical lesions include cyanosis of the
Figure 1. Showing severe congestion
1
comb
and
wattles
and
petechial
and edema of comb and wattles
haemorrhages on the hock. Also sudden
death occurs in a recumbent position and is preceded by pedaling movements and gasping. Some
birds show severe congestion of the comb, conjunctivitis and periorbital edema. Recovery is a
rare event.1
Aim of this project
Environmental factors may play a major role in both the primary as the secondary spread of
HPAI, especially in poultry slaughter- or collector houses (PCF) who receive poultry from
different origins and who are also distributing poultry on their turn. Therefore, the importance of
knowing the influence of environmental or other risk factors is that strategies can be designed to
improve the health of this poultry in order to decrease this potential transmission of pathogens to
commercial farms as well as to other poultry flocks.
The aim of this multi-intervention project was to investigate the incidence of HPAI and to
examine the most important causes of morbidity and mortality among poultry flocks located in
this district. In addition, risk factors which may contribute to the occurrence of disease were
identified.
H0 = There’s no relation between environmental factors and the spread of Avian Influenza in
poultry.
7
MATERIALS AND METHODS
Materials
The same surveillance had also been
conducted in January 2010, so general
information concerning the slaughterhouses
had already been gathered during that
period. Because of that a brief questionnaire
was used this time (appendix). The questions
in this new questionnaire concerned the
status of the environment (cleaning and
disinfection per consignment), the origin of
the poultry and the destination if this poultry
would be sold alive. The purpose was to find
out if environmental factors contribute in the
spread of avian influenza.
For the sample taking cotton swabs were used for both the environmental samples and the trachea
samples (figure 1). The swabs then were put in a tube with transport medium and kept at a
temperature of minus 20 degrees Celsius before they were transported to the laboratory in Cikole,
Lembang. In the lab the samples were kept at minus 70 degrees Celsius until they were
processed. During the sample taking protective clothing like an overall, boots, hand gloves and
mouth protection was used.
Figure 2. Taking a trachea sample 1
Methods
Social visits
The first stage of sample taking was to inform the six slaughterhouses in the area. So during the
first days the owners were visited to explain the purpose of the research project and they were
allowed to ask questions so that everything would be clear to them. The idea was that they called
the research team if a consignment was coming.
Because of the budget it was only possible to take samples from one consignment per
slaughterhouse per day.
The sample taking and data gathering
When an owner of a slaughterhouse expected a consignment that day, he would call the research
team so they could come to the place to take samples. With each consignment five environmental
swabs were first taken from the pen in which the poultry would be put. This was done by putting
a plastic bag over the boots, spraying distilled water on it, then walking around the pen and after
that it was possible to take swabs from the plastic bag underside the foot. The swabs were put in
medium transport and kept cool in a cool box while the team was still at the sample site.
In the mean time one of the Indonesian research members could fill in the questionnaire with the
owner or another respondent because native language was used for it.
After the consignment was unloaded five environmental swabs from the transport vehicle and
also five environmental swabs from the crates in which the chickens were transported were taken.
8
The newly arrived chickens were observed closely, because it was preferred to take samples from
the sick or moribund ones.
The number of trachea samples depended on the flock size. If the flock size was 50 chickens or
more an amount of 15 trachea swabs was taken. The exact prevalence of avian influenza in
Indonesia is unknown, but when looked at other studies with unvaccinated chicken you can
estimate a prevalence of 25%. If you take fifteen samples out of a population of 50 animals and
the estimated prevalence is 25% then the probability of diagnosing at least one animal as (truly)
positive is 99%. But if the prevalence is lower, like 15%, you still have a level of confidence of
95% with taking 15 trachea samples. This was calculated using Win Episcope 2.0.
Figure 3. Level of confidence with prevalence of 25%
If the flock size was smaller than 50 only ten trachea swabs were taken instead of the normal
amount of 15. If the flock size was less than 30 it was considered taking samples or not by
looking at the general health of the poultry. If for example the flock only consisted of nine
healthy looking chickens samples wouldn’t be taken, but it would be done if the same flock
consisted of nine dying chickens.
After the farm visits, precautions to avoid further spread of (possible) infection were taken as
much as possible. This was done by cleaning and disinfecting the boots and disposal of the waste.
Testing the field samples in the lab
After collecting the samples in the field, they could be kept at minus 20 degrees Celsius for a
maximum of 24 hours. That’s why they were taken to the laboratory in Cikole, Lembang as soon
as possible. In the lab the samples could be stored at minus 70 degrees Celsius for a longer
period. Before the samples were tested for the presence of Influenza A they were first pooled into
groups of five. The samples with a positive outcome were then tested for the specific H5
haemagglutinin of Influenza A.
In this laboratory, real-time PCR was used to test the field samples. The PCR provided an
extremely sensitive mean of amplifying a specific sequence of DNA or cDNA (because Influenza
viral genome is negative single-stranded RNA).1
9
The RNA was extracted using commercially available kits. RNA is an unstable molecule and
RNase (enzyme that degrades RNA) is virtually ubiquitous. So it was important to handle RNA
with disposable gloves and to use disposables such as pipette tips and reaction tubes that are
certified RNase-free.1
Questionnaire analysis
After translation from Indonesian back to English the questionnaires were analyzed at the
veterinary faculty in Institut Pertanian Bogor (IPB). For the analysis of the data the Statistical
Package for the Social Sciences (SPSS) version 13 was used with the Pearson Chi-square test for
the comparison of frequencies for significance.
10
RESULTS
Questionnaire analysis
General information concerning the slaughterhouses
Figure 4. Number of consignments
First we show the number of consignments that we took samples from. Each time with sample
taking a questionnaire was filled in. In the figure the consignment number and the percentage are
shown. The total number of samples taken is 60.
Sub-district
Village
Count
Count
%
%
16
27%
Anjatan
17
29%
Salamdarma
1
2%
Bojong
6
10%
Cikesik
1
2%
Cipunagara
13
22%
Kertasari
5
8%
Haurgeulis
11
18%
10
17%
Pagaden
1
2%
Haurkolot
6
10%
Jl. Cagak
2
3%
Manggungan
5
8%
Cijambe
1
2%
Sembung
1
2%
Unknown
9
14%
Padamulya
1
2%
Bunihayu
2
3%
Songgom
1
2%
Tanjung
2
3%
Unknown
9
14%
Bugis
Jati
11
District
Count
Province
%
Count
%
Indramayu
28
47%
West-Java
54
90%
Kuningan
2
3%
East-Java
1
2%
Purwakarta
7
12%
Unknown
5
8%
Pasuruan
1
2%
17
28%
5
8%
Subang
Unknown
Table 1. Origin of Poultry
Most poultry, namely 27%, is bought in Bugis village. Jati village is the second most used place
to buy chickens with 17%. But also a large number of 15% is unknown. There are also 5 villages
where poultry was bought only once. The most used sub-district for buying chickens is Anjatan,
with 28%. But also a relatively large number, 15%, is again unknown. Indramayu (47%) and
Subang (28%) are the most used districts to buy poultry. And almost all the poultry is bought in
West-Java (90%). Only 1 consignment (2%) was bought in East-Java. All these numbers are
shown in table 1 above.
Figure 5. Transport hygiene
In the above figure is shown that 73% of the transport vehicles were not cleaned before leaving
the PCF and 27% was cleaned before leaving. Disinfection of the transport did not happen in
96% of the occasions and did happen in 2%. The other 2% is unknown.
The transport crates were cleaned in 85% of all the occasions and the remaining 15% stayed
uncleaned before leaving the poultry collector facility. Disinfection of the crates almost shows
the same numbers: 15% is not disinfected, 83% is and the last 2% is unknown.
12
Specific information per slaughterhouse
PCF1
Count
Flocksize
17
Sum
Minimum
1055
43
Maximum
Mean
104
Std Deviation
62
20
Table 2. PCF1: Flock size
We took samples from 17 consignments in this slaughterhouse in Maranggi, Kosambi,
Cipunagara. The amount of samples depended on the flock size as is described in materials and
methods. The average flock size was 62 chickens, with a maximum flock size of 104 chickens
and a minimum of 43 chickens.
Figure 6. PCF1: Transport hygiene
In 59% of the occasions the transport was cleaned after the consignment arrived and 41% of the
occasions the transport vehicle was not cleaned before leaving the slaughterhouse. Only in 6% of
the occasions the transport was also disinfected, so 94% remained without disinfection.
The crates in which the chickens were transported were cleaned in 18% of the cases. Disinfection
happened in 24% of the occasions. In 82% of the occasions the transport crates weren’t cleaned
and in 76% of the occasions the crates were not disinfected before leaving the slaughterhouse.
Separate pen
Mixed with other poultry
Consignment
29%
71%
Cleaned
20%
-
Not cleaned
60%
-
Unknown
20%
-
Table 3. PCF1: Separating or mixing the poultry
In 29% of the total consigments the chickens were put in a separate pen. This separate pen was
cleaned in 20% of the cases, not cleaned in 60% of the occasions and for the other 20% it is
unknown if this separate pen was cleaned or not.
13
In 71% of the occasions the chickens from the consignments were mixed with other poultry that
was already present.
Village
Sub-district
District
Province
Bugis
Salamdarma
Anjatan
Indramayu
West-Java
Percent
94
6
100
100
100
Table 4. PCF1: Origin of the poultry
This owner always buys his chicken in the same sub-district, Anjatan.
The owner of this slaughterhouse didn’t sell live birds in 65% of the cases. In 35% of the
occasions live birds were sold. All of them in Kosambi village, Cipunagara sub-district.
PCF2
Count
Flocksize
7
Sum
2260
Minimum
40
Maximum
1080
Mean
323
Std Deviation
347
Table 5. PCF2: Flock size
In this slaughterhouse we took samples from 7 consignments. The average flock size was 323
chickens, with a maximum flock size of 1080 chickens and a minimum of 40 chickens.
Figure 7. PCF2: Transport hygiene
In 57% of the occasions the transport was cleaned before leaving the PCF, the other 43% was not
cleaned. The transport was not disinfected in 86% of the occasions and from 14% it is unknown
if the transport was disinfected or not. In 43% of the occasions the transport crates were cleaned
14
and 29% was disinfected too. 57% of the transport crates were not cleaned and not disinfected. In
14% of the occasions it is unknown if the transport crates were disinfected or not.
Consignment
71%
29%
Separate pen
Mixed with other poultry
Cleaned
80%
-
Not cleaned
20%
-
Unknown
0%
-
Table 6. PCF2: Separating or mixing the poultry
In 71% of the total consigments the chickens were put in a separate pen. This separate pen was
cleaned in 80% of the cases and not cleaned in 20% of the occasions. In 29% of the occasions the
chickens from the consignments were mixed with other poultry that was already present.
Village
Sub-district
District
Province
Percent
14
29
14
43
29
14
57
29
43
14
14
86
14
Cikesik
Kertasari
Jati
Unknown
Bojong
Cipunagara
Unknown
Kuningan
Purwakarta
Pasuruan
Subang
West-Java
East-Java
Table 7. PCF2: Origin of the poultry
This owner buys his chickens in many different places as you can see in table 7. Even 14% of the
consignments is coming from East-Java. In 29% of the occasions he sold live poultry, but for the
remaining 43% it is unknown.
PCF3
Flocksize
Count
11
Sum
762
Minimum
40
Maximum
141
Mean
76
Std Deviation
29
Table 8. PCF3: Flock size
In this slaughterhouse we took samples from 11 consignments. The average flock size was 76
chickens, with a maximum flock size of 141 chickens and a minimum of 40 chickens.
15
Figure 8. PCF3: Transport hygiene
The transport was not cleaned in 91% of the cases and not disinfected in 100% of the occasions.
18% of the crates were cleaned and disinfected. A number of 82% remained without cleaning and
disinfection.
Consignment
73%
27%
Separate pen
Mixed with other poultry
Cleaned
50%
-
Not cleaned
50%
-
Unknown
0%
-
Table 9. PCF3: Separating or mixing the poultry
In 73% of the occasions the chickens were put in a separate pen. This separate pen was cleaned in
50% of the cases and not cleaned in an other 50%. The remaining 27% of the chickens from the
consignments were mixed with other poultry that was already present.
Village
Sub-district
District
Province
Haurkolot
Manggungan
Haurgeulis
Indramayu
West-Java
Percent
54
45
100
100
100
Table 10. PCF3: Origin of the poultry
According to table 10, this owner buys his poultry always in the same sub-district. Most chickens
(64%) are then being slaughtered in the PCF and only 27% is sold alive in Sidamulya,
Cipunagara sub-district. A remaining 9% is unknown.
16
PCF4
Count
Flocksize
Sum
19
Minimum
1521
Maximum
35
Mean
125
Std Deviation
80
25
Table 11. PCF4: Flock size
In this slaughterhouse samples were taken from 19 consignments. The average flock size was 80
chickens, with a maximum flock size of 125 chickens and a minimum of 35 chickens.
The transport was never cleaned and disinfected, the transport crates were never cleaned either
and also not disinfected in 95% of the occasions. The remaining 5% is unknown.
Consignment
11%
89%
Separate pen
Mixed with other poultry
Cleaned
0%
-
Not cleaned
100%
-
Unknown
0%
-
Table 12. PCF4: Separating or mixing the poultry
Only 11% of the chickens from the consignments we sampled were put in a separate pen. This
separate pen was not cleaned in 100% of the cases. Most consignments (89%) were mixed with
other poultry as is shown in table 12.
Village
Kertasari
Percent
16
Jati
Sub-district
District
Province
32
Sembung
5
Padamulya
5
Bunihayu
11
Songgom
5
Unknown
26
Bojong
16
Cipunagara
37
Pagaden
5
Jl. Cagak
11
Cijambe
5
Unknown
26
Purwakarta
16
Subang
58
Unknown
26
West-Java
74
Unknown
26
Table 13. PCF4: Origin of the poultry
Above (see table 13) is shown were the poultry came from. No live birds were sold in 89% of the
cases whereas 10% is unknown.
17
PCF5
In this slaughterhouse in Salagedang, Jati, Cipunagara sub-district samples were only taken once.
The consignment came from the village of Jati, Cipunagara sub-district. The chickens were put in
a cleaned separate pen. The transport vehicle and the crates were not cleaned and disinfected
before leaving the PCF again. The chickens were not sold alive.
PCF6
Count
Flocksize
Sum
5
Minimum
280
Maximum
20
Mean
80
Std Deviation
56
33
Table 14. PCF6: Flock size
In this PCF 5 consignments were visited and sampled. The average flock size was 56 chickens,
with a maximum flock size of 80 chickens and a minimum of 20 chickens.
Figure 9. PCF6: Transport hygiene
Only 20% of the transport vehicles was cleaned and the transport was never disinfected. The
transport crates were cleaned and disinfected in 20% of all times.
Separate pen
Mixed with other poultry
Consignment
40%
60%
Cleaned
50%
-
Not cleaned
50%
-
Unknown
0%
-
Table 15. PCF6: Separating or mixing the poultry
The poultry was put in a separate pen in 40% of the occasions. This separate pen was cleaned in
50% of the cases and in 50% of the occasions it was not cleaned. The other 60% of the poultry
was mixed.
18
Percent
Village
Sub-district
District
Province
Jati
40
Tanjung
40
Unknown
20
Bojong
20
Cipunagara
80
Purwakarta
20
Subang
80
West-Java
100
Table 16. PCF6: Origin of the poultry
Most chickens (80%) were bought in Cipunagara sub-district, the other 20% was bought in
Bojong sub-district. See table 16.
In 60% of the occasions the poultry was sold alive and the other 40% was slaughtered in the PCF.
One third of the live sold chickens ended up in Julang village, Pagaden sub-district, one third was
sold in Sukamandi village and one third was sold in Jati, Cipunagara sub-district. That is all in
Subang district, West-Java.
Laboratory results
Three out of six PCF’s had positive tests for Influenza A in the trachea swabs and/or in the pens.
All the crates and transport vehicles had a negative test result. All PCF’s were tested negative for
the presence of H5. Note, the 5% positive pens from PCF6 were part of the consignments which
also had positive test outcome for the trachea. So only a total of 13% of all the consignments had
positive test results.
M-PCR
PCF
Trachea
Pens
Crates
Transport
H5-PCR
1
1 (2%)
0
0
0
0
2
0
0
0
0
0
3
0
1 (2%)
0
0
0
4
0
0
0
0
0
5
0
1 (2%)
0
0
0
6
4 (7%)
3 (5 %)
0
0
0
Table 17. Positive PCR Results per PCF
The test results were compared with the answers from the questionnaires with the use of the
Pearson Chi-square test. A significant relation between two variables is proofed when the P-value
of the test is 0,05 or less.
No significant relations were found between any of the aspects that were asked questions about in
the questionnaire and being tested positively for Influenza A in the crates or pens (P > 0.05).
Also, because all the crates, transport vehicles en the H5-PCR tests were negative it is not
19
possible to show any relation between the results of the questionnaires and the laboratory tests
results. This means there is no significant relation between these results.
20
DISCUSSION
PCF locations
Six poultry collector facilities were visited during this research project. The locations are shown
on the maps in appendix II. Three of the six poultry collector facilities (PCF2, 5 and 6) were
located close to each other on the same road. That is why it was expected that there would be
more transmission of disease between these facilities because of human involvement in
secondary spread.1,5,6,8-16 When we take a closer look to the poultry collector facilities which had
a positive Influenza A test outcome it is seen that 9% of the positive test cases belong to PCF5
and 6 (table 17) which are located at close distance from each other. But PCF2 had no positive
tests for Influenza A and the other positive cases (PCF1 and 3) are all in very different locations.
No explanation can be given.
Origin of Poultry
With every consignment that was sampled questions were asked about the origin of the poultry.
This was to see if there is a connection between infected flocks and the place they come from.
Although there is no significant relationship between being tested positive for Influenza A and
the place of origin, we see there is a lot of indirect contact possible between the collector
facilities. PCF2, 4, 5 and 6 all buy chickens from Jati, Cipunagara. Both PCF2 and 4 also buy
their chickens in Kertasari village. But from PCF4 a lot of information about the origin of the
poultry is missing because some of these questions were not answered in the questionnaires. But
also with the questionnaires of the other poultry collector facilities the village origin often
proofed to be difficult to trace. So maybe there is even a higher chance of indirect contact
between facilities than we know of.
Especially PCF2 and 4 have their poultry from a lot of different origins and even from far away
(PCF2 from East-Java). According to a study of identifying risk factors of HPAI movement of
poultry is a significant risk factor for the spread of HPAI.16 So a higher risk possibility can be
expected for these slaughterhouses. And because some slaughterhouses buy their poultry from
PCF2, it is expected that these slaughterhouses have a higher risk factor too. Note, it was not
asked in the questionnaire if the slaughterhouses buy their poultry from another PCF and it was
not recorded so no numbers are available, but it was told by the respondents from the collector
facilities that this happens sometimes.
Destination of sold live poultry
One of the questions in the questionnaire was about the selling of live birds because one of the
goals of this research project was to look at the risk factors in spreading Avian Influenza and the
contribution of the poultry collector facilities in this. But nothing can be said about this possible
spread for no samples were taken from the destinations of the sold live birds so no laboratory test
results are available.
Transport vehicles
The transport vehicles were sampled right after the consignment had been unloaded. In this way
it was possible to take swabs from right under the place where the poultry was placed upon the
vehicle. The thought behind this was that these were the places with most faeces pollution in
which the virus could be contained. Also, it was tried to take samples from the wheels and from
21
both sides of the vehicle. For every transport type it was tried to do it as much the same as
possible. But with more than one sample-taking team it is hard to say if it worked in the field,
because sometimes new people were helping. Questions about cleaning and disinfecting the
transport vehicles were asked and the responses showed that in most cases there was no cleaning
and disinfection at all. But not a single pool of swabs from the transport vehicles was tested
positive for Influenza A. This is not in line with the expectations of this research project for it
was thought that these kind of environmental factors would play an important role in the spread
of HPAI. As is said before, secondary spread of disease is possible and especially mechanically
after contact with contaminated faeces. Also, five consignments had a positive test for the trachea
samples and the transport vehicles of these consignments were cleaned (but not disinfected) in
only one occasion and the other four remained uncleaned and not disinfected, so there were at
least some expectations about these specific transport vehicles being tested positive. No
explanation can be given for these opposite results.
Transport crates
The same can be said about the transport crates. In only one occasion had the crate been cleaned
and disinfected and the crates of the remaining four consignments were not. But still, no crate
was tested positive for the presence of Influenza A.
Pens
A total of 9% of all the pens have been found positive for Influenza A. When we take a closer
look at these positive cases it is interesting to see that PCF3 and PCF5 both had a positive test
from samples that were taken on the same day with the same enumerator and that the
consignment had been put in a separate and cleaned pen (table 9 and results PCF5) which is
remarkable (although cleaning is not the same as disinfection). And that the day before this the
same enumerator had visited PCF6 which had a positive test for the trachea swabs and the pens
that day. And two days before that this PCF6 had positive trachea tests (but not the pens then).
This of course can be coincidence and it is not possible to proof it (and no significant relation was
found), but it has to be kept in mind that transmission because of human involvement is one of
the possibilities.
The pens were sampled before the consignment was unloaded, but maybe it would have been
interesting to take samples 24 hours later too. Also, some poultry collector facilities had more
than one pen, for example PCF6 had two pens, but it was not marked on the questionnaire in
which of the pens the poultry was put. So that may be another explanation for the test results
being positive one time and negative the other time.
Problems in the field
It was a quiet period for the slaughterhouses and as can be seen in figure 4 it differs even among
the slaughterhouses. But some slaughterhouses received more consignments than were actually
tested because these consignments were not unloaded in the pens but were sold again
immediately. Therefore it is possible it looked quieter than what is was in reality.
Also the data from the slaughterhouse PCF4 which received the most consignments (19) is
probably more reliable than the data from PCF5 which only had one consignment. In this way it
is difficult to compare slaughterhouses with each other.
22
There were problems with filling in the questionnaires so a lot of data is missing and unknown.
For example, one of the questions was about how many consignments a PCF received between
our visits but this question has not been answered or wrongly answered (how many chickens
instead of consignments) in 95% of the occasions. Two of sixty questionnaires were filled in a
couple of weeks afterwards so the level of confidence can be regarded as unreliable.
Even the sample gathering was not done consistently. Before starting the fieldwork it had been
decided that only one consignment per PCF per day would be sampled but at PCF2 three
consignments were sampled on the same day.
It proofed to be difficult to clean and disinfect boots and protective clothing when leaving a
slaughterhouse before proceeding to the next slaughterhouse. When visiting a PCF our transport
was parked on the terrain of the PCF, often right next to the pen in which the poultry had been
put.
According to a study done in the Netherlands, it is possible that a person can spread disease
mechanically on the same day or even longer if the person becomes infected themself. This
happened the most with visits right after infection of a farm. The animals on these farms are
likely not yet symptomatic, and therefore escaping diagnosis. So with this in mind it should be
considered if the research team acted as a possible vector of disease transmission. And although
such events may be difficult to prevent, awareness of this possibility is very important to keep
this from happening.15
23
CONCLUSION AND RECOMMENDATIONS
According to this study, cleaning and disinfection of transport vehicles and transport crates and
the place of origin of poultry does not make any difference in the spread of Avian Influenza in
this research area. Also no H5 subtype of Avian Influenza was found, so this highly pathogenic
form does not seem to cause problems in this area although mutation from circulating lower
subtypes of Avian Influenza into the highly pathogenic variant is a possibility. Even with these
results it is always important to take preventive measures to avoid transmission and secondary
spread of disease.
24
REFERENCES
1) I. CAPUA, D.J. ALEXANDER (2009) Avian Influenza and Newcastle Disease: a field
and laboratory manual. Springer, pp. 1-11,32-34,39-43
2) D.E. SWAYNE, D.L. SUAREZ (2000) Highly pathogenic avian influenza. Rev Sci Tech,
Vol. 19, pp. 463-82
3) S. DAVISON, R.J. ECKROADE, A.F. ZIEGLER (2003) A review of the 1996-98
nonpathogenic H7N2 avian influenza outbreak in Pennsylvania. Avian Dis, Vol. 47,
pp.823-827
4) D.A. SENNE, D.L. SUAREZ, D.E. STALLNECHT, J.C. PEDERSEN et al. (2006)
Ecology and epidemiology of avian influenza in North and South America. Dev Biol, Vol.
124, pp. 37-44
5) D.J. ALEXANDER (2000) A review of avian influenza in different bird species.
Veterinary Microbiology, Vol. 74, pp. 3-13
6) L.D. SIMS, J. DOMENECH, C. BENIGNO, S. KAHN, A. KAMATA, J. LUBROTH, V.
MARTIN, P.ROEDER (2005) Origin and evolution of highly pathogenic H5N1 avian
influenza in Asia. Veterinary Record, Vol. 157, pp. 159-164
7) D.A. SENNE (2007) Avian influenza in North and South America, 2002-2005. Avian Dis,
Vol. 51, pp. 167-173
8) W. UTTERBACK (1984) Update on avian influenza through February 21, 1984 in
Pennsylvania and Virginia. Proceedings of the 33rd Western Poultry Disease Conference.
9) S.E. GLASS, S.A. NAQI, L.C. GRUMBLES (1981) Isolation of avian influenza virus in
Texas. Avian Dis, Vol. 25, pp. 545-549
10) P.J. HOMME, B.C. EASTERDAY, D.P. ANDERSON (1970) Avian influenza virus
infections, II, Experimental epizootiology of influenza A-turkey-Wisconsin-1966 virus in
turkeys. Avian Dis, Vol. 14, pp. 240-247
11) R.J.H. WELLS (1963) An outbreak of fowl plague in turkeys. Vet Rec, Vol. 75, pp. 783786
12) D.C. JOHNSON (1984) AI task force veterinarian offers practical suggestions. Broiler
Indust, Vol. 47, pp. 58-59
13) L.J. KING (1984) How APHIS “war room” mobilized to fight AI. Broiler Indust, Vol. 47,
pp. 44-51
14) I. CAPUA, F.MUTTONELLI (2001) An atlas and text on avian influenza. Papi Editore,
pp. 1-236
15) D.E. TE BEEST, J.A. STEGEMAN, Y.M. MULDER, M. VAN BOVEN, M.P.G.
KOOPMANS (2010) Exposure of Uninfected Poultry Farms to HPAI (H7N7) Virus by
Professionals During Outbreak Control Activities. Zoonoses and Public Health, pp. 1-7
16) L. LOTHA, M. GILBERT, J. WUD, C. CZARNECKIE, M. HIDAYAT, X. XIAOE
(2011) Identifying risk factors of highly pathogenic avian influenza (H5N1 subtype) in
Indonesia. Preventive Veterinary Medicine, Vol. 102, Issue 1, pp. 50-58
25
APPENDIX I
QUESTIONNAIRE – PCF/ PSH
SURVEILLANCE OF PCF and PSH
Identification no PCF/PSH:
Date :
Enumerator :
Information of PCF/PSH
1) Name of owner:………………….…………………………………………………….
2) Name of respondent: ...………………………………………………………………..
3) Address of PCF/PSH
Sub Village :………………………………………………………………………….
Village
:…………………………………………………………………………..
Sub District :………………………………………………………………………….
4) GPS:
Lat
:……………………………………………………………………………….....
Long :……………………………………………………………………………….....
26
QUESTIONNAIRE – PCF/ PSH
Questions for each individual consignment
PCF/PSH ID no.:
Consignment ID no.:
Date :
Enumerator :
1) Type of poultry:
□ Broiler
□ Ayam kampung
□ Spent parent stock □ Male layers
□ Spent layers
□ Other ……………………. ......
2) Number of birds:……………………………………………………………………........
3) Origin of poultry (Village / sub-district / district / province):
Village……………………………………………………………………………………
Sub-district………………………………………………………………………………
District……………………………………………………………………………………
Province…………………………………………………………………………………
4) Was the consignment put into a separate pen or mixed with other poultry?
□ Separate pen*
□ Mixed
□ Don’t know
*If put in a separate pen, was the pen clean?
□ Yes
□ No
□ Don’t know
5) Method of transportation:
□ Truck
□ Car
□ Motorcycle
□ Other ……………………. .....
6) Was the transport cleaned before leaving the PCF/PSH?
□ Yes
□ No
□ Don’t know
7) Was the transport disinfected before leaving the PCF/PSH?
□ Yes
□ No
□ Don’t know
27
8) Were the crates that the birds were transported in cleaned before leaving the PCF/PSH?
□ Yes
□ No
□ Don’t know
9) Were the crates that the birds were transported in disinfected before leaving the
PCF/PSH?
□ Yes
□ No
□ Don’t know
□
10) What was the destination of any sold live birds:
No live birds sold
Village……………………………………………………………………………………
Sub-district………………………………………………………………………………
District……………………………………………………………………………………
Province…………………………………………………………………………………
11) How many consignments has the PCF/PSH received since our previous visit?
..………………………………………………………………………………………….
To be completed by the enumerator:
12) Estimated number of dead birds in this consignment:
...............………………………………………………………………………………….
13) Number of samples taken from this consignment:
……………………………………………………………………………………............
28
Appendix II
Research area; Cipunagara sub-district, Subang, West Java
Closer view from the image above
29