Faculty of Veterinary Medicine IPB. District Livestock Services of Subang. Collaboration:

Transcription

1 Avian Influenza Surveillance in Chicken and Nomadic Duck Flocks in Cipunagara, Subang Multi-Intervention Pilot Project on the control and prevention of HPAI in Cipunagara sub-district in Kabupaten Subang November 2011 Collaboration: Faculty of Veterinary Medicine IPB District Livestock Services of Subang

2 Avian Influenza Surveillance of Chicken and Nomadic Duck Flocks in Cipunagara, Subang 1 INTRODUCTION By all accounts, Highly Pathogenic Avian Influenza (HPAI) continues to pose a considerable disease burden for the poultry industry in Indonesia. However, in the absence of adequate surveillance programs, reliable measures on the frequency of HPAI occurrence within the different poultry sectors are lacking, with the possible exception of Sector IV. The Indonesian Dutch Partnership on HPAI (IDP) and the Dinas Peternakan of Subang district and West Java province have been implementing a multi-intervention pilot project on the control and prevention of HPAI. The aim of this project was to try to control HPAI in one small area of West Java using a variety of intervention measures. The chosen area was Cipunagara, which is a subdistrict of Subang district in West Java. As part of this multi-intervention project, surveillance was carried out designed to detect the presence of AI virus in farmed poultry in Cipunagara. Specifically, this surveillance program had the following objectives: To detect the presence of AI virus or the exposure to AI virus in poultry flocks which are residing in Cipunagara during a specified time period To investigate the potential role of the different sectors of the poultry industry to harbour and transmit AI virus To investigate the movements of nomadic duck flocks in, out, and within Cipunegara subdistrict during the rice harvesting period To collect and store AI viruses for future isolation and characterization The surveillance was conducted during paddy harvesting time periods, because it is at this time that nomadic duck flocks are most frequently moved into the subdistrict. The inclusion of this poultry species in the surveillance activities was seen as of paramount importance because of the role of nomadic duck flocks in the epidemiology of HPAI which was observed elsewhere. In addition, surveillance was carried out during two separate time periods during the year to take account of possible seasonal fluctuations in the occurrence of HPAI. INTRODUCTION 1

3 Avian Influenza Surveillance of Chicken and Nomadic Duck Flocks in Cipunagara, Subang 2 MATERIALS AND METHODS 2.1 Study area & population The surveillance of AI in duck and chicken flocks was conducted in Cipunagara subdistrict. This subdistrict is situated in the central-eastern part of Subang District of West Java. It has an approximate area of hectares. Based on data from a previous study (Sudarnika et al., 2010), combined with more recent data held by the Dinas Peternakan of Subang district, there were eight commercial breeding farms, 25 broiler farms and six operational poultry collecting facilities/slaughterhouses (PCF/PSH) located in Cipunagara at the time of the surveillance. During and after rice harvest times, nomadic duck flocks move frequently into and out of the area to graze the rice paddies. It was estimated that approximately 50 duck flocks would be present in Cipunagara during the rice harvesting time. 2.2 Time The surveillance was carried out during two separate time periods which coincided with the rice harvests. The first surveillance period (hereafter called surveillance period 1) was conducted from the 15 th of April until the 30 th of June The second surveillance period (surveillance period 2) started on the 23 rd of November and ended on the 23rd of January. Surveillance of broiler-breeder flocks was carried out as flocks were depopulated and took place from September 2010 until April During surveillance period 2, broiler farms were surveyed as part of a syndrome surveillance which started in October 2010 and lasted until June Data Collection Data collected from the different poultry flocks consisted of tracheal and/or cloacal swabs and/or blood samples and is detailed for each poultry type in Table 1. Additional information was collected via questionnaires. MATERIALS AND METHODS 3

4 Avian Influenza Surveillance of Chicken and Nomadic Duck Flocks in Cipunagara, Subang Table 1: Type of samples and number of samples collected from poultry during a HPAI surveillance program in Cipunagara, Subang. Poultry type (sampling unit) Sample type Sample size (min max) a Nomadic ducks (flock) Tracheal swabs Cloacal swabs Blood Broilers Tracheal swabs 15 (flock) Broiler-breeders (flock) PCF/PSH (consignment) a Tracheal swabs 15 Blood 15 Tracheal swabs Environmental swabs b 15 Sample size of nomadic duck flocks and PCF/PSH consignments was dependent on flock/consignment size. Sample size calculations are described in section b Environmental swabs were only taken during surveillance period Sample size Nomadic duck flocks The sampling unit was the duck flock. A group of ducks was considered a flock if it arrived at and departed from a rice field as one group, i.e if it was transported as one group. The number of samples taken from a nomadic duck flock was based on the ability to detect a positive sample in a flock with a minimum expected prevalence of 5%, a test sensitivity of 95%, a test specificity of 100% and a 95% confidence level. Calculated sample sizes were dependent on flock size and ranged from a minimum of 10 samples (flock size equals 10) to a maximum of 60 samples (flock size equal to and larger than 400). Required sample sizes for different flock sizes were tabulated for easy use in the field. Sample sizes were adjusted upwards to accommodate for the fact that they were combined into pooled samples of five swabs each (for example, if the calculated sample size was 58 samples, 60 samples were taken in order to make 12 pooled samples). To simplify the study design and consequent field work, blood samples were taken from the same ducks from which swab samples were collected. The number of collected blood samples would be sufficient to estimate a prevalence of MATERIALS AND METHODS 4

5 Avian Influenza Surveillance of Chicken and Nomadic Duck Flocks in Cipunagara, Subang 25% of sero-positive birds with a 10% precision and a 95% confidence level. However, during surveillance period 2 it was decided to collect a standard number of 10 blood samples per flock. The reason for this was that during surveillance period 1 a lot of resistance was met from the duck owners with regard to taking blood samples as it was a widely shared belief that this would impair egg production Broiler flocks The flock was the sampling unit. A group of broilers was considered a flock if they were raised in the same house. Sample size was based on the ability to detect an infected flock with a minimum expected within-flock prevalence of 20%, a test sensitivity of 95%, a test specificity of 100% and a confidence level of 95%. Assuming an average flock size of 1000 birds or higher, the calculated sample size was 15 tracheal swabs per flock Broiler-breeder flocks Again, the flock was the sampling unit with the definition of a flock being a group of broiler-breeder birds kept in one house. Sample size considerations were identical to those for broiler flocks meaning that 15 tracheal swabs were taken per flock. In addition, 15 blood samples were collected from the same birds from each flock PCFs and PSHs For sampling within PCFs and PSHs, the sampling unit was a poultry consignment. The definition of a poultry consignment was a batch of birds arriving on the same transport vehicle (i.e. truck, car or motorcycle). During surveillance period 1, sample size was calculated based on the same criteria as those used for broiler flocks and broiler-breeder flocks and amounted to a sample size of 15 swabs. During surveillance period 2, consignment size was also taken into consideration. Ten samples were taken from consignments with less than 50 birds whereas 15 samples were taken from consignments with 50 birds or more. In MATERIALS AND METHODS 5

6 Avian Influenza Surveillance of Chicken and Nomadic Duck Flocks in Cipunagara, Subang addition, during surveillance period 2, 15 environmental swabs were taken from the PCF/PSH whenever a poultry consignment was sampled Sampling strategy Sampling of nomadic duck flocks Nomadic duck flocks which were present within Cipunagara at the start of the surveillance period were sampled as soon as possible after the start of the study. Thereafter, all flocks were sampled which entered the subdistrict. During surveillance period 1, flocks which left the subdistrict were sampled again, provided the time period between the two sampling dates was longer than three weeks. During surveillance period 2, nomadic duck flocks were sampled only once. Animals were not sampled at random but as they were presented to the sampling officers by the herdsmen or flock owners. It was attempted to preferentially sample sick ducks. From each selected animal, a tracheal and a cloacal sample was collected. It was attempted to also take a blood sample from each selected animal but this was not always permitted by the owner or herdsmen. Two to 2.5 ml blood was collected from the brachial vein, stored in a serum tube and transported in a cool box to a field station Sampling of broiler flocks During surveillance period 1, flocks were sampled twice. The first sampling took place upon arrival on the farm as day-old chicks (DOCs) whereas the second sampling took place when broilers were harvested for slaughter. Birds to be sampled were selected in a non-random manner. During surveillance period 2, flocks were only sampled when there was flock mortality of 1% or higher during two consecutive days. In this case, sick or recently dead animals were sampled. This syndrome surveillance was part of larger project in which trained Dinas personnel served as biosecurity advisors to broiler farmers, involving regular farm visits and pre-harvest blood sampling. MATERIALS AND METHODS 6

7 Avian Influenza Surveillance of Chicken and Nomadic Duck Flocks in Cipunagara, Subang Sampling of broiler-breeder flocks Flocks were sampled at end of lay, before going to slaughter. Sampling was carried out by farm personnel who were only occasionally supervised by the Dinas Peternakan farm entry restrictions. Random selection of birds can therefore not be guaranteed. Blood (2-2.5ml) was collected from the brachial vein and transported in a cool box to a field station for further processing Sampling of poultry consignments coming to PCFs/PSHs Consignments of poultry to PCFs/PSHs were sampled on the day of their arrival. A maximum of one consignment per PCF/PSH per day was sampled. During surveillance period 1, flocks that originated from Cipunagara and which were sent to PCFs/PSHs in Cipunagara were sampled only once (on the farm). Sick or recently dead animals were preferentially sampled; if they were not present in the consignment, healthy birds were randomly selected. During surveillance period 2, five environmental swabs were taken from the holding pen in which the consignment was to be placed by walking with plastic overshoes through the pen and collecting the material which had stuck on the plastic on a swab and repeating this five times. After unloading had finished, five swabs each were taken from the transport vehicle and from the transport crates Data Collection by Questionnaire Questionnaires were used to collect additional data on the sampled poultry flock or poultry consignment. Respondents were farm/flock owners or workers and PCF/PSH owners or workers. All questionnaires were administered by trained enumerators. Information pertaining to nomadic duck flocks, included flock characteristics such as flock size, breed, age, production type (i.e. egg, meat, dual purpose), movement records (where from, where to, date of movement, method of movement) and history of vaccination. Broiler flock information which was collected included general farm characteristics, origin of the DOCs, flock size, total mortality during the production period, slaughter age and slaughter destination. MATERIALS AND METHODS 7

8 Avian Influenza Surveillance of Chicken and Nomadic Duck Flocks in Cipunagara, Subang Information collected from broiler-breeder flocks included company name, flock size, poultry brand, age and total mortality during the production cycle. In the PCFs/PSHs, information was collected regarding general PCF/PSH characteristics and more specific information about the consignment such as poultry type, size, origin and transportation method. All questionnaires are listed in Appendix A Laboratory Analysis Swab samples were transported in a cool box to the laboratory on the same day that they were collected. In the laboratory, tracheal, cloacal and environmental swab samples were combined into pools of five swabs each. Pooled samples were analyzed for the presence of Influenza A genomic material using a real-time M-PCR. Positive pooled samples with the M-PCR were tested further using a real-time H5-PCR. All PCR analyses were conducted at the Cikole Laboratory of the Livestock services of West Java province. In the field station, serum was separated from whole blood within 12 hours of sample taking, stored in an eppendorf tube and kept at -20⁰C until its transport in a cool box to the Cikole laboratory. All serological tests were conducted at this laboratory. Serum samples were analyzed for the presence of antibodies against Influenza A virus using an Elisa test. Samples with a positive titre were further tested for the presence of antibodies against H5 antigen (obtained from BBalitvet) using a Haemagglutination Inhibition (HI) test as described in the OIE Terrestrial Manual (OIE, 2005). Titres equal to or greater than 2 4 were considered positive. 2.5 Data Analysis Data collected by questionnaire was entered directly into a Microsoft Access database and analyzed descriptively by generating frequency distributions of variables of interest. Laboratory results were entered into an Excel spreadsheet. All data analysis was performed using SPSS version MATERIALS AND METHODS 8

9 Avian influenza surveillance of chicken and nomadic duck flocks in Cipunagara, Subang 3 Results 3.1 Nomadic ducks Nomadic duck samples During surveillance period 1, a total of 59 nomadic flocks were sampled which included nine flocks which were sampled for the second time when they were about to leave Cipunagara subdistrict. Fifty flocks were sampled during surveillance period 2 which did not include any flocks which were sampled twice. The total number of collected samples and the average number of samples per flock are shown in Table 1. During surveillance period 1, the collection of serum samples was met with a lot of resistance from the side of the duck flock owners who were afraid of losses in egg production. Only 46 flock owners gave permission for the collection of serum samples and the full targeted sample size was collected from only one flock. As a consequence, for the second surveillance period the decision was made to only take 10 serum samples per flock. Table 1 Number of sampled nomadic duck flocks and number of collected samples during two surveillance periods in Cipunagara, Subang Surveillance period No. of sampled flocks No. of collected samples Average no. of collected samples per flock Cloaca Trachea Serum Cloaca Trachea Serum RESULTS 9

10 Avian influenza surveillance of chicken and nomadic duck flocks in Cipunagara, Subang Origin of the duck flocks All the duck flocks which were sampled during the two surveillance periods were based in West Java Province when they were not moving around. The majority of the flocks during both surveillance periods were based in Subang district followed by Indramayu district (Figure 1). During the first surveillance period, duck flocks which originated from Subang district, came from the sub-districts Cipunagara (n=16), Compreng (n=5), Pamanaukan (n=45), Pusakanagara (n=3), Sagalaherang (n=3) and Sarangpanjang (n=1). During surveillance period 2, duck flocks within Subang originated from the sub-districts Cipunagara (n=14), Binong (n=11), Pusaka Jaya (n=5), Tambak Dahan (n=5), Subang (n=2), Compreng (n=1) and Legok Kulon (n=1). Figure 1 Districts of origin of nomadic duck flocks sampled during two surveillance periods in Cipunagara, Subang. \ RESULTS 10

11 Avian influenza surveillance of chicken and nomadic duck flocks in Cipunagara, Subang Characteristic of nomadic duck flocks Average size of the sampled duck flocks was 261 birds (range ) in surveillance period 1 and 288 birds (range ) in surveillance period 2. The majority of duck flocks were raised for the purpose of egg production whereas only a few flocks were raised for meat (Figure 2). Figure 2 Production purposes of nomadic duck flocks sampled during two surveillance periods in Cipunagara, Subang Duck flocks consisted mainly of Java ducks although especially during surveillance period 1, a large proportion of farmers did not know what the breed was of their ducks (Figure 3). RESULTS 11

12 Avian influenza surveillance of chicken and nomadic duck flocks in Cipunagara, Subang Figure 3 Type of breeds found in nomadic duck flocks sampled during two surveillance periods in Cipunagara, Subang Information on how the ducks were housed at night during their stay in Cipunagara was only collected during the second surveillance period. All the duck flocks were confined in cages or pens at night and the majority of these flocks (n=44; 88%), were confined on the rice fields which they grazed during the day. The remaining six flocks would be taken to a location away from the rice fields. In general, the farmers did not vaccinate their duck flocks against any diseases. During the first surveillance period only one flock had been vaccinated against AI and ND and this had happened more than one year ago. During the second surveillance period, two duck flocks had been vaccinated against AI of which one flock was vaccinated during the last three months and of which one flock the date of vaccination was unknown Movement of nomadic duck flocks Forty-three flocks left Cipunagara during surveillance period 1 after staying in Cipunagara for an average of 23 days (range 5-90 days) of which 26 flocks remained in Subang district, 15 flocks moved to the neighbouring district of Indramayu and for two flocks the destination was unknown. During the second surveillance period, 44 flocks planned to leave Cipunagara after staying an average of 19 days (range 2-90 days). Twenty-seven of these flocks remained in Subang, four RESULTS 12

13 Avian influenza surveillance of chicken and nomadic duck flocks in Cipunagara, Subang flocks moved to Indramayu, one flock to Cirebon and six flocks planned to move to an unknown destination. Information on transportation methods for the duck flocks was only collected during the second surveillance period. The most common method for transporting the duck flocks into, within or out of Cipunagara was by pick-up truck (Figure4). Figure 4 Methods of transport of nomadic duck flocks into Cipunagara, within Cipunagara and out of Cipunagara during the second surveillance period Laboratory analysis The results of the M-PCR analysis of the pooled swab samples taken from nomadic duck flocks are shown in Table 2. During the first surveillance period, 21 (42%) nomadic duck flocks that were present in or entered Cipunagara had at least one pooled sample test positive for Influenza A. whereas 2 (22%) flocks which were sampled when they left the district had one or more positive pooled samples. One of this two flocks was negative at the first sampling. During surveillance period 2, 30 (60%) flocks were found positive. The infection was more frequently detected in tracheal swab samples during surveillance period 1 whereas in surveillance period 2 pooled samples of cloacal swabs where more frequently positive. None of the RESULTS 13

14 Avian influenza surveillance of chicken and nomadic duck flocks in Cipunagara, Subang samples which tested positive with the M- PCR gave a positive test result with the H5 PCR. Table 2: Number of positive pooled swab samples and number of positive duck flocks on the basis of M-PCR test results during two surveillance periods in Cipunagara, Subang. Flocks with at least one positive pooled sample were considered positive. Pooled swab samples No. of positive pooled samples (%) Surveillance period 1 No. of positive flocks (%) Tracheal swabs only 32 (6.3%) 5 (10.0%) Cloacal swabs only 25 (4.9%) 8 (16.0%) Both tracheal and cloacal swabs 7 (1.4%) 8 (16.0%) Total 71 (14%) 21 (42.0%) Surveillance period 2 Tracheal swabs only 27 (5.1%) 6 (12.0) Cloacal swabs only 45 (8.6%) 9 (18.0%) Both tracheal and cloacal swabs 28 (5.3%) 15 (30.0%) Total 128 (24.4%) 30 (60.0%) Serological test results of duck serum are presented in Table 3. The seroprevalence of antibodies against Influenza A as measured by an Elisa test was above 80% during both surveillance periods resulting in a flock prevalence of 89.1% and 100% during the first and second surveillance period respectively. The analysis for H5 antibodies with the HI test resulted in a sero-prevalence of 1.7 % and 0.6% for first and second surveillance periods respectively. This meant that 15.2%of the flocks in the first surveillance period and 6% of the flocks in the second surveillance period had one or more samples with antibodies against H5. RESULTS 14

15 Avian influenza surveillance of chicken and nomadic duck flocks in Cipunagara, Subang Table 3: Number of positive serum samples and number of positive duck flocks based on the Elisa and Haemagglutination Inhibition (HI) tests during two surveillance periods in Cipunagara, Subang Elisa HI Surveillance period 1 Surveillance period 2 No. of positive samples (%) 508 (84.8%) 10 (1.7%) No. of positive flocks (%) 41 (89.1%) 7 (15.2%) No. of positive samples (%) 428 (85.6%) 3 (0.6%) No. of positive flocks 50 (100%) 3 (6%) 3.3 Poultry Collector Facilities (PCFs)/Poultry Slaughter Houses (PSHs) Samples collected in PCFs/PSHs The total number of poultry consignments which were sampled and the total number of samples which were collected during the two surveillance periods are presented in Table 4. The PCFs/PSHs which were sampled during the two surveillance periods were the same except for one PCF/PSH (Dimiyati was replaced by Idrus). Almost twice as many consignments were sampled during the first surveillance period as during the second surveillance period. On average, PCFs/PSHs received 2.5 consignments per week (range 1-3) during surveillance period 1 and 1.25 consignments per week (range 1-3) during surveillance period 2. For the six PCFs/PSHs in Cipunagara, this meant that a mean total of 15 consignments were delivered per week during surveillance period 1 and 7.5 consignments per week during surveillance period 2. During the first surveillance period, a consignment consisted of an average of 122 birds (range ) whereas during the second surveillance the average consignment size was 101 birds (range ). During surveillance period 2 only, 900 environmental samples were collected from pens, crates and transport vehicles (pick-up trucks, motorcycles etc.). RESULTS 15

16 Avian influenza surveillance of chicken and nomadic duck flocks in Cipunagara, Subang Table 4 Number of sampled consignments and collected samples from PCFs/PSHs during two surveillance periods in Cipunagara, Subang Name No. of consignments Average number of birds per consignment Min.-max. number of birds per consignment No. of collected samples No. of collected environmental samples Surveillance period 1 Dayat Maranggi Karja H. Sika Dayat Jati Tarda Dimiyati Total Surveillance period 2 Dayat Maranggi Karja H. Sika Dayat Jati Tarda Idrus Total Types of Poultry Consignments There were two types of poultry that were slaughtered and/or sold by the PCF/PSHs during the two surveillance periods, namely broiler and parent stock. The majority of consignments were broilers (n=100 or 85% in the first period and n=48, or 80% in the second period of surveillance) Origin and Transport of Poultry Consignments Most consignments which came into Cipunagara were from West Java Province (n=112 or 96% in the first surveillance and n=54 or 90% in the second surveillance). The other consignments sampled during the first surveillance period were from Banten (n=3, 3%), East Java (n=1, 1%) and Yogyakarta (n=1, 1%), while during the second surveillance period one consignment (2%) originated from East RESULTS 16

17 Avian influenza surveillance of chicken and nomadic duck flocks in Cipunagara, Subang Java and five consignments (8%) had an unidentified place of origin. The districts of origin of the poultry consignments are presented in Figure 5. During both surveillance periods, the majority of consignments originated from Subang District and the neighboring Indramayu District. Some consignment (n=5) in two PCFs/PSHs were sold live birds after they arrive at the PCF. The destination of live birds after they arrive at PCFs were Subang district (4 consigment) and Karawang district (1 consigment). The consignments in the first surveillance period were transported to the PCFs/PSHs by pick-up truck (n=48, 41%), motorcycle (n=45, 49%) or truck (n=24, 21%). Similarly, most of the consignments during the second surveillance period were transported to the PCFs/PSHs using pick-up trucks (n=27, 45 %), followed by the use of motorcycles (n=22, 37%) and regular trucks (n=11, 18%). Figure 5 Origin of poultry consignments (by district) delivered to PCFs/PSHs during two surveillance periods in Cipunagara, Subang RESULTS 17

18 Avian influenza surveillance of chicken and nomadic duck flocks in Cipunagara, Subang Laboratory analysis of samples collected at PCFs/PSHs The 1755 tracheal swabs which were collected during the first surveillance period were combined into 351 pooled samples. One pooled sample (0.9%) tested positive with the M-PCR indicating the presence of Influenza A genetic material but subsequently tested negative for the presence of H5 antigen. Of the 174 pooled tracheal swab samples which were collected during the second surveillance period, eight pooled samples (4.6%) collected from four consignments (6.7%) tested positive for Influenza A but were negative with the H5 PCR. The 900 samples collected from the PCF/PSH environment and transport equipment were combined into 180 pooled samples of which five pooled samples (2.8%) of five consignments (8.3%) tested positive for Influenza A but all tested negative in the H5 PCR Broiler farms Sampling of broiler farms During the first surveillance period, 21 DOC flocks and 25 broiler flocks going for slaughter were sampled, resulting in 63 and 75 pooled tracheal swab samples respectively. During this surveillance period, there was a disease outbreak associated with high mortality on one broiler farm in Cipunagara. In addition to swab samples taken on this farm, 15 tracheal samples were collected from an adjacent backyard chicken flock which also experienced mortality. During the second surveillance period, 15 tracheal samples were taken from one broiler farm on two separate occasions after the flocks experienced mortalities exceeding 1% during two subsequent days Origin and destination of broiler flocks Information on the origin and destination of the sampled broiler flocks was only collected during the first surveillance period. Ten of the 21 DOC flocks which were sampled originated from Subang district whereas two flocks came from DKI Jakarta. The origin of the remaining 9 flocks was unknown; farmers could only state the name of the company as the DOC source. Seven DOC flocks which were hatched RESULTS 18

19 Avian influenza surveillance of chicken and nomadic duck flocks in Cipunagara, Subang in Subang came from the subdistrict of Pagaden whereas two flocks came from Cipunagara itself. The originating subdistrict of the 10 th flock hatched in Subang was unknown. The majority of the broiler flocks produced on farms in Cipunagara were slaughtered in West Java (n=23, 92%) while the two other broiler farmers involved in the surveillance did not know where their broilers would be slaughtered. The districts within West Java where the broiler flocks were destined to be slaughtered is shown in Figure 6. The majority of flocks were slaughtered in Subang. Within Subang, the broiler flocks were sent to be slaughtered in the subdistrict of Pagaden (n=10), Cipunagara (n=3), Binong (n=1) and an unknown subdistrict (n=1). Figure 6 The slaughter destination within West Java Province of broiler flocks produced in Cipunagara, Subang during the first surveillance period Laboratory analysis of broiler flock samples The RT-PCR analysis results of DOC and broiler flock pooled tracheal swab samples are presented in Table 5. Trachea swabs samples were taken from one backyard (sector 4) flock because it experienced high mortality and it was located close to the broiler farm in which a suspected HPAI event occurred. During the RESULTS 19

20 Avian influenza surveillance of chicken and nomadic duck flocks in Cipunagara, Subang second surveillance period, the two flocks that tested positive for H5 came from the same farm on two separate occasions. Table 5 The results of PCR analysis on tracheal swab samples collected from broiler farms in Cipunagara Subang and sector 4 Source of tracheal swab sample Number of flocks sampled Number of collected Positive pooled samples Positive Flocks pooled samples M-PCR H5 PCR M-PCR H5 PCR Surveillance period 1 DOC Broiler Sector Surveillance period 2 Broiler Sector 1 & 2 farms From May 2010 until April 2011, five breeder farms culled their (grand)- parent stock and sent them for slaughter. A total of 92 flocks from these farms were sampled. Neither the M-PCR nor the H5 PCR analysis resulted in any positive results. The results of the serological analysis are shown in Table 6. RESULTS 20

21 Avian influenza surveillance of chicken and nomadic duck flocks in Cipunagara, Subang Table 6 Serological test results of 92 broiler-breeder flocks at the time of culling in Cipunagara, Subang Breeder Farm Sampling month Number of sampled flocks ELISA (%) Seroprevalence HI 2 4 (%) Mean HI titer ± S.D. A September (100) 362 (67%) 3.56±1.92 B October ND 60 (100%) 7.07±0.84 C January ND 164 (91%) 6.37±2.27 D January ND 179 (99%) 8.00±1.33 E April ND 417 (99%) 7.58 ± 1.42 ND: Not done RESULTS 21

22 Avian Influenza surveillance of chicken and nomadic duck flocks in Cipunagara, Subang 4 DISCUSSION Disease monitoring can be described as the ongoing activities to assess the health and disease status of a given population. Disease surveillance on the other hand, implies a more active system whereby some sort of action is taken if the collected data indicates that the disease occurrence exceeds a certain threshold (Salman, 2003). Therefore, the data collection activities which are described in this report cannot be classified as an AI surveillance system because they lacked any sort of follow-up in response to the AI cases which were detected. With the exception of the Participatory Disease Surveillance & Response (PDS/R) program, surveillance programs designed for the detection and monitoring of AI infections in poultry in Indonesia are virtually absent. The PDS/R program, although commendable in its scope and ambition, suffers from some obvious shortcomings (Perry et al., 2009). First, it is almost exclusively focused on the detection of HPAI events in backyard poultry flocks (Sector 4) and therefore misses out on the occurrence of HPAI in commercial poultry flocks. Secondly, the PDS/R program cannot provide valid prevalence or incidence data because of the lack of a random sampling approach among other forms of bias. And thirdly, it doesn t give any information on the occurrence of subclinical H5N1 infections in chickens and ducks because the surveillance relies entirely on the detection of clinical disease, confirmed by the use of a rapid antigen test. The present monitoring program was part of a larger multi-intervention pilot project taking place in the subdistrict of Cipunagara in Subang. The monitoring tried to address some of the major criticisms levied against the PDS/R program. Thus, it targeted primarily the commercial poultry sectors, as a seroprevalence survey of some important poultry diseases occurring in backyard poultry flocks (including AI) had already been conducted previously as part of the multi-intervention pilot (IPB, 2010). Because duck flocks are known to be able to harbor the AI virus subclinically, serum DISCUSSION 22 22

23 Avian Influenza surveillance of chicken and nomadic duck flocks in Cipunagara, Subang samples were taken in this species in order to demonstrate possible subclinical infections or exposure to disease. During the two surveillance periods, HPAI was detected in two broiler flocks and in an adjacent poultry flock in Sector 4. In between the two surveillance periods, in August 2010, a third broiler flock was found positive for HPAI. Three cases over a 15 month period could well be an underestimate as much of it was dependent on reporting by the farmer. Except for one case in a Sector 4 backyard flock, in which sector no routine monitoring took place, HPAI was not detected in any other poultry sector. Based on this limited amount of data, it would suggest that farms in Sector 3 play an important role in the epidemiology of H5N1, at least in Cipunagara. Ducks have been implicated as silent carriers and shedders of H5N1 virus (Hulse-Post et al., 2005) and grazing duck flocks have been spatially associated with HPAI outbreaks in chickens in Thailand (Gilbert et al., 2006) and Vietnam (Pfeiffer et al., 2007). In Cipunagara, the role of nomadic duck flocks in the occurrence of HPAI outbreaks remains in some doubt as none of the sampled duck flocks residing in Cipunagara during the two surveillance periods tested positive for shedding H5 virus although 15% (n=7) of the flocks in the first surveillance period and 6% (n=3) of the flocks in the second surveillance period had positive antibody titres against H5. As only one flock during surveillance period 1 and two flocks during surveillance period 2 were vaccinated against AI, it would suggest that positive titres found in birds of the remaining flocks were due to exposure to field virus. However, we have no evidence that these antibody titres are directed against a highly pathogenic avian influenza (HPAI) as no neuraminidase subtype was determined. Cross-reactions in the HI test resulting in a positive titre against H5 subtype have also not been excluded. Studies of ducks carried out elsewhere in Indonesia did demonstrate the presence of H5 virus albeit at low levels. A survey of 194 duck farms in two districts of Banten and four districts of West Java found that 2.1% of farms tested positive with the H5 PCR and 31.4% of farms had at least one H5 positive serum sample (FAO, 2011). Similarly, a year-long longitudinal study in four districts of Central Java whereby 96 farms where visited on seven separate occasions detected H5 virus DISCUSSION 23 23

24 Avian Influenza surveillance of chicken and nomadic duck flocks in Cipunagara, Subang in 2.5% of the duck flock visits and at least one H5 sero-positive bird in 19.5% of the duck flock visits (Henning et al., 2010). On the basis of these results, the authors of both studies concluded that although exposure of nomadic duck flocks to H5 virus in Indonesia is a fairly frequent occurrence, actual virus shedding appears to be very low. In contrast, exposure to and shedding of other Influenza A viruses was found to be high in this survey; 42% of the flocks during the first surveillance period and 60% of the flocks during the second surveillance period had one or more birds shedding Influenza A virus. This differs somewhat with results from the study of FAO (2010) where 15% of the duck flocks had one or more pooled swab sample test positive with the M-PCR. This difference can be partly explained by the fact that the study by the FAO only took tracheal swab samples whereas our study also collected cloacal swab samples. An additional 16% of flocks during the first surveillance period and 18% of flocks during the second surveillance period were detected by the use of cloacal samples. In general, low-pathogenic AI viruses (LPAI) in waterfowl have an affinity for the intestinal tract and are therefore more likely to be detected with cloacal swab samples although there are some strain-dependent exceptions (Spackman et al., 2008). More pooled tracheal swab samples were positive with the M-PCR than pooled cloacal swab samples during the first surveillance period but the situation was reversed during the second surveillance period. Because we are dealing with pooled samples, it is impossible to determine if there were more birds shedding virus through the respiratory tract than through the intestinal tract or visa versa. It seems advisable therefore, if costs allow, that future surveillance studies of AI in ducks collect both tracheal as well as cloacal swabs in order to increase the sensitivity of the survey. Although it remains possible that ducks are implicated in the spread of HPAI virus in Indonesia, they do not appear to disseminate the virus over large distances. This follows from the movement data collected during this study, which demonstrated that about two-thirds of the flocks remained in Subang District during the two surveillance periods while almost all of the other flocks moved only as far as DISCUSSION 24 24

25 Avian Influenza surveillance of chicken and nomadic duck flocks in Cipunagara, Subang a neighboring district. Movement of duck flocks to other provinces appears to be uncommon as is corroborated by previous studies (CIVAS, 2006; FAO, 2010) Poultry collection facilities (PCF), are a gathering place for birds from different regions before being slaughtered or sold into the market. Studies in DKI Jakarta have demonstrated that a high proportion of these PCFs have evidence for the presence of HPAI virus (CIVAS, 2008) which can be partly explained by HPAI infected poultry consignments delivered to the PCFs (CIVAS, 2009 & 2011). It is not inconceivable that these PCFs serve as transmissions hubs for AI virus whereby cross-contamination takes place of poultry, vehicles and equipment entering the PCF which is subsequently disseminated to other collector houses, slaughterhouses and farms. Furthermore, there are important public health issues associated with these PCFs because of the intensive contacts that take place between poultry and humans and the unsafe disposal of waste products into the environment surrounding the PCF. However, our surveillance conducted over two separate time periods in six PCFs in Cipunagara could not demonstrate the presence of HPAI virus in incoming poultry consignments or in selected areas of the PCF environment. This would suggest that the HPAI infection pressure in PCFs in Cipunagara is likely to be low. In contrast to poultry consignments transported to PCFs in DKI Jakarta (CIVAS 2009 & 2011), poultry consignments delivered to PCFs in Cipunagara generally did not come from far away as the majority of transports originated from places within Subang District or from neighboring Indramayu District and only few transports originated from places outside of West Java Province. From a disease and animal welfare point of view this is a desirable situation and we can only hope that the results of our data collection over a relatively short period of time reflect the true situation in Cipunagara. Sector 1&2 farms in Indonesia generally have higher levels of biosecurity and disease control than farms in Sector 3. Presumably, these types of farms have fewer outbreaks of HPAI than farms in the other poultry sectors although anecdotal reports are plenty regarding HPAI events on breeder farms, including on a farm in Cipunagara. The collection of tracheal swab samples and blood samples was not DISCUSSION 25 25

26 Avian Influenza surveillance of chicken and nomadic duck flocks in Cipunagara, Subang carried out under our control due to the strict biosecurity regimes existing on these farms and possible also because of an element of secrecy towards outsiders which is part of the management of these farms. None of the 92 breeder flocks which were sampled in the course of this surveillance program tested positive with either the M- PCR or the H5-PCR. However, because of our lack of control of the sample collection process, it cannot be excluded that there were flocks during this period of time with clinical signs compatible with HPAI which were not sampled for our program. Serological analysis of the blood samples resulted in flock seroprevalences in excess of 90% with the exception of breeder farm A. Although positive titres ( 2 4 ) are no direct proof of flock protection, it does suggest the presence of vaccination programs with adequate coverage. Conclusions On the basis of our surveillance studies we can conclude the following: - HPAI was only detected in 3 broiler flocks and in an adjacent village poultry flock. It emphasizes the fact that sector 3 farms are vulnerable because biosecurity standards are low and vaccination against AI is not routinely practiced. - There is no evidence that nomadic duck flocks play a role in the epidemiology of HPAI in Cipunagara. However, longitudinal surveillance studies carried out over extended periods of time are needed to confirm this. - There is evidence for the presence of other Influenza A virus in nomadic duck flocks. Further virus isolation and sequencing is required to elucidate the nature of these viruses. - The majority of movements of poultry, including nomadic duck flocks, consignments to PCFs/PSHs, and broiler flocks going to slaughter, travelled relatively short distances. The poultry sectors in Cipunagara therefore, play only a limited role in virus transmission over large distances. DISCUSSION 26 26