Epidemiology study and risk assessments of highly pathogenic avian influenza H5N1 in free flying birds in Thailand by Jarunee Siengsanan-Lamont D.V.M, MVS (Conservation Medicine) School of Veterinary and Biomedical Sciences Faculty of Health Sciences Murdoch University Western Australia ―This thesis is presented for the degree of Doctor of Philosophy of Murdoch University‖ 2010
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Epidemiology study and risk assessments of highly pathogenic avian
influenza H5N1 in free flying birds in Thailand
by
Jarunee Siengsanan-Lamont
D.V.M, MVS (Conservation Medicine)
School of Veterinary and Biomedical Sciences
Faculty of Health Sciences
Murdoch University
Western Australia
―This thesis is presented for the degree of Doctor of Philosophy
of Murdoch University‖
2010
I declare that this thesis is my own account of my research and contains as its main content
work which has not previously been submitted for a degree at any tertiary education
Pothieng, Parntep Ratanakorn. Investigation of HPAI H5N1 serological and virological
status of free flying birds in Banglane district, Thailand. 12th International Symposium on
Veterinary Epidemiology & Economics (ISVEE) 10-14 August 2009. Durban, South Africa
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Acknowledgments I would like to thanks my Siengsanan and Lamont families; Charoon, Pranee, Parin, Penny,
and Owen who were always my big supporters. Thank you for all my supervisors (Ian
Robertson, Stuart Blacksell, Trevor Ellis, Stan Fenwick, and Kristine Warren), John and Peta
Edwards, friends, and colleagues who had contributed to this work one way or another.
Many thanks for all the staff at the Monitoring and Surveillance Center for Zoonotic diseases
in Wildlife and Exotic Animals at the Faculty of Veterinary Science, Mahidol University and
Wildlife research unit, Department of National Parks, Wildlife, and Plant Conservation who
had a big contribution in the field and laboratory work.
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Table of contents
ABSTRACT ........................................................................................................................................................ I
PUBLICATIONS ............................................................................................................................................ IV
ACKNOWLEDGMENTS .............................................................................................................................. VI
TABLE OF CONTENTS .............................................................................................................................. VII
LIST OF TABLE .............................................................................................................................................. X
LIST OF FIGURES ....................................................................................................................................... XII
1.2.1 Nature of the virus and its replication .............................................................................................. 2 1.2.2 Evolution of the avian influenza (H5N1) virus ................................................................................. 6
1.3 EPIDEMIOLOGY OF HPAI H5N1 VIRUS ................................................................................................... 10 1.3.1 Mode of transmission ...................................................................................................................... 10 1.3.2 The spread of the disease and its molecular epidemiology ........................................................... 10 1.3.2 Host range in wild birds ................................................................................................................. 16 1.3.3 Persistence of the virus in the environment .................................................................................... 16 1.3.4 The role of wild birds in the persistence or transmission of H5N1 ............................................... 17
1.4 CLINICAL FINDINGS AND PATHOLOGY OF H5N1 HPAI DISEASE ............................................................ 17 1.5 IMPACTS OF HPAI OUTBREAKS ............................................................................................................... 20 1.6 OUTBREAKS OF HPAI H5N1 IN THAILAND ............................................................................................. 22 1.7 SURVEILLANCE PROGRAM FOR HPAI H5N1 VIRUS IN WILD BIRDS IN THAILAND .................................. 26 1.8 BACKGROUND AND DEVELOPMENT OF THE CURRENT PROJECT .............................................................. 27
MATERIALS AND METHODS ................................................................................................................... 32
2.1 STUDY DESIGN ......................................................................................................................................... 32 2.2 STUDY SITE SELECTION ............................................................................................................................ 33
2.2.1 Mixed habitat types for wild birds and domestic poultry ............................................................... 33 2.2.2 The study area and its history of H5N1 HPAI poultry outbreaks .................................................. 34
2.3 RETROSPECTIVE STUDIES ......................................................................................................................... 36 2.3.1 Existing analysis of data on an Avian Influenza (H5N1) surveillance program in wild birds in
Thailand (2004-2007) .............................................................................................................................. 36 2.3.2 Questionnaire study ........................................................................................................................ 37
2.4 PROSPECTIVE STUDIES ............................................................................................................................. 38 2.4.1 Serological and virological surveillance program for HPAI H5N1 virus and molecular study in
wild birds (2007 and 2008) ...................................................................................................................... 38 2.4.2 Observational study ........................................................................................................................ 39 2.4.3 Risk assessments ............................................................................................................................. 40
2.5 COLLECTION OF FIELD SAMPLES .............................................................................................................. 40 2.5.1 Selection of wild bird species and surveillance within the study site ............................................ 40 2.5.2 Sample techniques and procedures; collection of swabs and blood ............................................. 41 2.5.3 Recording of field sample data ....................................................................................................... 42 2.5.4 Safety procedure ............................................................................................................................. 42
2.6 LABORATORY DIAGNOSIS ........................................................................................................................ 43 2.6.1 Necropsy procedure ........................................................................................................................ 45 2.6.2 Viral culture .................................................................................................................................... 45 2.6.3 Haemagglutination test (HA) .......................................................................................................... 46 2.6.4 Multiplex Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) ................................... 47 2.6.5 Gene sequencing: ............................................................................................................................ 48 The procedure for gene sequencing is described in Chapter 5 (Section 5.2). ........................................ 48 2.6.6 Microneutralisation test (NT test) .................................................................................................. 48
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2.6.6.1 Serum treatment ........................................................................................................................................ 49 2.6.6.2 Virus antibody reaction ............................................................................................................................. 49 2.6.6.3 MDCK cells culture .................................................................................................................................. 49 2.6.6.4 Back titration ............................................................................................................................................. 50 2.6.6.5 Control set ................................................................................................................................................. 50 2.6.6.6 End point determination; CPE based NT assay ........................................................................................ 50
COMPARISON OF OUTBREAKS OF H5N1 HIGHLY PATHOGENIC AVIAN INFLUENZA IN
WILD BIRDS AND POULTRY IN THAILAND ........................................................................................ 52
3.1 INTRODUCTION ........................................................................................................................................ 52 3.2 MATERIAL AND METHODS....................................................................................................................... 53
3.2.1 Collection of field samples .............................................................................................................. 53 3.2.2 Virus isolation and identification ................................................................................................... 54 3.2.3 Statistical analysis ........................................................................................................................... 57
STUDY OF RISK FACTORS FOR HPAI H5N1 INFECTION IN SMALL POULTRY FARMS
USING A QUESTIONNAIRE SURVEY ...................................................................................................... 67
4.1 INTRODUCTION ........................................................................................................................................ 67 4.2 MATERIALS AND METHODS ..................................................................................................................... 67
4.2.1 Questionnaire design and trial ....................................................................................................... 67 4.2.2 Study site design and plan .............................................................................................................. 68 4.2.3 Field questionnaire study ................................................................................................................ 69 4.2.4 Data analysis ................................................................................................................................... 70
5.3 RESULTS ................................................................................................................................................. 134 5.3.1 Results of virus isolation and serological testing of wild birds at the study site ......................... 134 5.3.2 Results of genetic characterization and phylogenetic analysis ................................................... 140
OBSERVATIONAL STUDY TO INVESTIGATE INTERACTIONS OF WILD BIRDS WITH
POULTRY SPECIES .................................................................................................................................... 168
6.1 INTRODUCTION ...................................................................................................................................... 168 6.2 MATERIALS AND METHODS ................................................................................................................... 169
6.2.1 Study design .................................................................................................................................. 169 6.2.2 Observation site selection ............................................................................................................. 170 6.2.3 Observation field data collection .................................................................................................. 177 6.2.4 Statistical analysis ......................................................................................................................... 178
7.3.4.1 Probability of wild birds being infected with H5N1 (p1) ...................................................................... 234 7.3.4.2 Probability of subclinical infection with sufficient virus shedding to cause infection (p2) .................. 237 7.3.4.3 Probability of the presence of wild birds in poultry keeping areas (p3) ................................................ 247 7.3.4.4 Probability of wild birds being in close proximity to and/or feeding together with domestic poultry (p4)
7.3.5.1 Probability of lesser whistling ducks infected with HPAI H5N1 virus (p1) ......................................... 250 7.3.5.2 Probability of subclinical infection with shedding sufficient virus to cause infection in poultry (p2).. 251 7.3.5.3 Probability of lesser whistling ducks present in farms (p3) ................................................................... 253 7.3.5.4 Probability of lesser whistling ducks living close to and/or feeding together with domestic poultry (p4)
GENERAL DISCUSSION AND CONCLUSIONS ................................................................................... 264
8.1 INTRODUCTION ...................................................................................................................................... 264 8.2 GENERAL DISCUSSION AND CONCLUSIONS ............................................................................................ 264 8.3 LIMITATIONS OF THE PROJECT AND REQUIREMENTS FOR FURTHER STUDY ........................................... 267
APPENDIX I .................................................................................................................................................. 270
APPENDIX II ................................................................................................................................................. 271
APPENDIX III ............................................................................................................................................... 273
TABLE 2.1; DESCRIPTION OF CPE SCORING .......................................................................................... 51 TABLE 3.1; POSITIVE WILD BIRD FAMILIES AND THEIR HEALTH STATUS IN THE WILD BIRD
SURVEILLANCE DURING 2004-2007 ................................................................................................ 60 TABLE 4.1; TIMETABLE OF THE FIELD QUESTIONNAIRE STUDY ................................................... 71 TABLE 4.2; SUMMARY OF RESPONDENTS’ GENERAL INFORMATION .......................................... 73 TABLE 4.3; TYPES AND NUMBER OF ANIMALS KEPT BY VILLAGERS .......................................... 74 TABLE 4.4; FOOD GIVEN TO POULTRY IN THE SURVEYED HOUSEHOLDS .................................. 76 TABLE 4.5; SOURCE OF WATER USED FOR POULTRY ........................................................................ 77 TABLE 4.6; COMPARISON OF SELLING PROCEDURES AND TYPES OF POULTRY SOLD ........... 79 TABLE 4.7; AGES OF POULTRY TRADED AND NUMBER PER TRADE ............................................. 82 TABLE 4.8; SOURCES OF NEW POULTRY ................................................................................................ 83 TABLE 4.9; MEASURES IMPLEMENTED TO ENSURE NEW BIRDS WERE FREE FROM DISEASE
.................................................................................................................................................................. 84 TABLE 4.10; QUARANTINE PERIOD .......................................................................................................... 85 TABLE 4.11; FREQUENCY AND NUMBER OF EGGS TRADED ............................................................ 85 TABLE 4.12; MANAGEMENT OF MANURE AND FREQUENCY OF CLEANING WITH DIFFERENT
POULTRY TYPES .................................................................................................................................. 86 TABLE 4.13; FREQUENCY AND PERCENTAGE OF VILLAGERS SEEING A RANGE OF WILD
BIRDS EVERY DAY IN BACKYARDS, FARMS OR PADDY FIELDS (N=217) .......................... 89 TABLE 4.14; FREQUENCY AND PERCENTAGE OF VILLAGERS SEEING A RANGE OF WILD
BIRDS LESS FREQUENTLY THAN ONCE A DAY IN BACKYARDS, FARMS OR PADDY
FIELDS (N=217) ..................................................................................................................................... 91 TABLE 4.15; VILLAGERS’ ACTIONS IF THEY SUSPECTED THEIR BIRDS HAD AVIAN
INFLUENZA (N=239)* .......................................................................................................................... 93 TABLE 4.16; MEASURES CONSIDERED BY VILLAGERS AS NECESSARY TO
PREVENT/CONTROL AVIAN INFLUENZA (N=239) ...................................................................... 94 TABLE 4.17; METHODS VILLAGERS WERE USING TO PROTECT THEIR BIRDS FROM AVIAN
INFLUENZA (N=239) ............................................................................................................................ 94 TABLE 4.18; METHODS ADOPTED BY VILLAGERS TO PREVENT MEMBERS OF THEIR
HOUSEHOLDS FROM BECOMING INFECTED WITH AVIAN INFLUENZA (N=239).............. 95 TABLE 4.19; SOURCES OF INFORMATION ABOUT AVIAN INFLUENZA FOR VILLAGERS
(N=239) .................................................................................................................................................... 96 TABLE 4.20; HISTORY OF HPAI H5N1OUTBREAK ................................................................................. 98 TABLE 4.21; TYPES OF POULTRY AND HPAI OUTBREAK HISTORY ................................................ 99 TABLE 4.22; THE FLOCK SIZE AND INFLUENCE ON A HISTORY OF AI .......................................... 99 TABLE 4.23; RISK FACTORS FOR THE PRESENCE OF AN OUTBREAK IN A HOUSEHOLD/FARM
(N = 217) ................................................................................................................................................ 100 SHADING INDICATED CATEGORY WITH P ≤ 0.25............................................................................... 122 TABLE 4.24; VARIABLES INCLUDED IN THE FINAL LOGISTIC REGRESSION MODEL* ........... 123 TABLE 5.1; COMMON NAME AND SPECIES OF WILD BIRDS SAMPLED IN THE SURVEY ....... 136 TABLE 5.2; SEROPREVALENCE TO H5N1 VIRUS FOR WILD BIRDS TESTED IN THE SURVEY138 TABLE 5.3; PREVALENCE OF VIRAL ISOLATION IN THE SURVEY ................................................ 138 TABLE 5.4; COMPARISON OF AMINO ACID SEQUENCES OF HAEMAGGLUTININ (HA) GENES
................................................................................................................................................................ 160 TABLE 5.5; COMPARISON OF AMINO ACID SEQUENCES OF NEURAMINIDASE (NA) GENES 161 TABLE 6.1; OBSERVED SITES AND TIME PERIODS IN ONE SEASON (FOUR MONTHS) ............ 170 TABLE 6.2; AVERAGE TEMPERATURE AND HUMIDITY AT THE STUDY SITES IN EACH TIME
PERIOD (T1 – T8) ................................................................................................................................. 179 TABLE 6.3; FREQUENCY OF WILD BIRDS OBSERVED AND PERCENTAGE OF THEM BEING
OBSERVED ........................................................................................................................................... 180 TABLE 6.4; SUMMARY OF TOTAL OBSERVED NUMBERS (TN) AND FREQUENCY OBSERVED
(FO) OF WILD BIRDS AT THE STUDY SITES ............................................................................... 184 TABLE 6.5; SUMMARY OF TOTAL OBSERVED NUMBERS (TN) AND FREQUENCY OBSERVED
(FO) OF WILD BIRDS AT THE STUDY SITES B2, D2 AND C1 ................................................... 187 TABLE 6.6; NUMBERS OF WILD BIRDS SEEN AND THEIR BEHAVIOURS AT THE WILD BIRD
ROOSTING /NESTING AREAS (SITES A1 AND C1) ..................................................................... 192 TABLE 6.7; NUMBERS OF OBSERVED WILD BIRDS AND THEIR BEHAVIOURS AT THE POND
AND RICE PADDY FIELD SITES (A2 AND C2) ............................................................................. 198
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TABLE 6.8; NUMBERS OF OBSERVED WILD BIRDS AND THEIR BEHAVIOURS IN THE
BACKYARD AREAS (SITE B1 AND D1) ......................................................................................... 204 TABLE 6.9; NUMBERS OF OBSERVED WILD BIRDS AND THEIR BEHAVIOURS AT THE OPEN
SYSTEM DUCK FARMS (SITE B2 AND D2)................................................................................... 210 TABLE 7.1; DEFINITION OF SCORES USED IN THE QUALITATIVE ASSESSMENT ..................... 231 MODIFIED FROM PFEIFFER ET AL. (2006) ............................................................................................. 231 TABLE 7.2; DEFINITION OF CATEGORIES FOR UNCERTAINTY FOR QUALITATIVE
ASSESSMENT ...................................................................................................................................... 231 (PFEIFFER ET AL., 2006) .............................................................................................................................. 231 TABLE 7.3; RISK RANKING MATRIX [MODIFIED FROM (VOSE, 2008)] ......................................... 232 TABLE 7.4; DISTRIBUTIONS USED IN @RISK® IN THE QUANTITATIVE RISK ASSESSMENT 233 TABLE 7.5; THE RELATIVE PATHOGENIC EFFECTS OF INOCULATING AVIAN INFLUENZA
VIRUS INTO DIFFERENT WILD BIRDS ......................................................................................... 240 TABLE 7.6; ASSESSMENT OF THE PROBABILITY OF SUBCLINICAL INFECTION WITH
SHEDDING OF SUFFICIENT VIRUS TO CAUSE INFECTION (P2) [*IF B=N/A, B=1] ............ 245 TABLE 7.7; ASSESSMENTS OF P3 AND P4 ............................................................................................. 249 TABLE 7.8; QUALITATIVE RISK ASSESSMENT OF AN INFECTED WILD BIRD SHEDDING AN
INFECTIOUS DOSE OF VIRUS CLOSE TO POULTRY ................................................................. 249
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List of Figures
FIGURE 1.1; STRUCTURE OF INFLUENZA VIRUS TYPE A ..................................................................... 5 FIGURE 1.2; STEPS OF THE VIRAL REPLICATION IN A HOST CELL ................................................... 5 FIGURE 1.3; MAP SHOWING AREAS REPORTING CONFIRMED AVIAN INFLUENZA H5N1
CASES IN POULTRY AND WILD BIRDS SINCE 2003 .................................................................... 14 FIGURE 1.4; PHYLOGENETIC TREE OF AVIAN INFLUENZA H5N1 VIRUSES ISOLATED
BETWEEN 2004 AND 2006 .................................................................................................................. 15 FIGURE 1.4; EFFECT OF HPAI H5N1 ON THAI POULTRY EXPORTING TRADE .............................. 22 FIGURE 1.5; DISTRIBUTION OF HPAI H5N1 OUTBREAK CASES (RED DOTS) IN POULTRY IN
THAILAND (2004 – 2006) ..................................................................................................................... 25 FIGURE 1.6; THE STUDY PROCESS ............................................................................................................ 30 FIGURE 2.1; LOCATION OF BANGLANE DISTRICT IN THAILAND .................................................... 35 FIGURE 2.2; SATELLITE IMAGE OF THE STUDY SITE IN BANGLANE SUBDISTRICT,
BANGLANE DISTRICT SHOWS RICE PADDY FIELDS AND VILLAGES .................................. 36 FIGURE 2.3; FLOW CHART OF LABORATORY PROCESS FOR AVIAN INFLUENZA H5N1 VIRAL
DETECTION ........................................................................................................................................... 44 FIGURE 2.4; DIAGRAM OF NT TEST PLATE ............................................................................................ 51 FIGURE 3.1; THAI PROVINCES WHERE DOMESTIC POULTRY OUTBREAK CASES WERE
REPORTED AND WILD BIRD SURVEILLANCE CONDUCTED (MODIFIED MAP;
THANAPONGTHARM AND NOIMOH, 2006) ................................................................................... 62 FIGURE 4.1; FREQUENCY OF SELLING POULTRY PER YEAR ............................................................ 78 FIGURE 4.2; FREQUENCY OF PURCHASING POULTRY PER YEAR ................................................... 82 FIGURE 4.3; FACTORS CONSIDERED BY VILLAGERS TO INCREASE THE RISK OF
INTRODUCING AVIAN INFLUENZA TO THEIR POULTRY (N=239) ......................................... 93 FIGURE 4.5; HOUSING OF BREEDERS AND ADULT COCKS IN A COMMERCIAL COCK
FIGHTING FARM ................................................................................................................................ 127 FIGURE 4.6; HOUSING OF JUVENILE BIRDS IN A COMMERCIAL COCK FIGHTING FARM ...... 128 FIGURE 4.7; A COCK FIGHTING COMPETITION ................................................................................... 128 FIGURE 5.1; NUMBER OF SAMPLES COLLECTED IN THE STUDY DURING FEBRUARY 2007 TO
OCTOBER 2008 AND THE TIMING OF SEROLOGICAL AND VIROLOGICAL POSITIVE
SAMPLES .............................................................................................................................................. 139 FIGURE 5.2; COMPLETE NUCLEOTIDE SEQUENCES OF HA GENES (A) AND NA GENES (B) OF
WVM/07 AND APS/07 VIRUSES ....................................................................................................... 142 FIGURE 5.3; ALIGNMENT OF AMINO ACID SEQUENCES OF HAEMAGGLUTININ (HA) GENE 155 FIGURE 5.4; PHYLOGENETIC TREE OF NUCLEOTIDE SEQUENCES OF THE HA GENE OF THAI
ISOLATES AND ISOLATES FROM THE WILD BIRD SURVEILLANCE ................................... 158 FIGURE 5.5; PHYLOGENETIC TREE OF NUCLEOTIDE SEQUENCES OF NA GENE OF THAI
ISOLATES AND ISOLATES FROM THE WILD BIRD SURVEILLANCE ................................... 159 FIGURE 5.6; MAP OF THAILAND SHOWS LOCATIONS WHERE THE POULTRY OUTBREAK IN
PICHIT PROVINCE AND THE POSITIVE WILD BIRDS WERE DETECTED ............................ 166 FIGURE 6.1; ABANDONED BUILDING WHERE PIGEONS WERE NESTING (SITE A1) ................. 172 FIGURE 6.2; RICE PADDY FIELDS AND A NATURAL POND (SITE A2) ........................................... 172 FIGURE 6.3; BACKYARD AREAS BEHIND ACCOMMODATION FOR FACTORY WORKERS (SITE
B1) .......................................................................................................................................................... 173 FIGURE 6.4; AN OPEN SYSTEM DUCK FARM IN A SLAUGHTER HOUSE (SITE B2) .................... 173 FIGURE 6.5; ASIAN OPEN BILL STORK ROOSTING SITE (SITE C1) ................................................. 175 FIGURE 6.6; ABANDONED LOTUS POND NEXT TO RICE PADDY FIELDS (SITE C2) .................. 176 FIGURE 6.7; A HOUSEHOLD WITH BACKYARD POULTRY AND PIGS (SITE D1) ........................ 176 FIGURE 6.8; AN OPEN SYSTEM LAYER DUCK FARM (SITE D2) ...................................................... 177 FIGURES 6.9; COMPARISON OF BEHAVIOURS AND ACTIVITIES OF WILD BIRDS OBSERVED
AT THE SITES ...................................................................................................................................... 213 FIGURES 6.9 AND 6.10; WATER BIRDS AND TERRESTRIAL BIRDS SHARING A RICE PADDY
FIELD ..................................................................................................................................................... 216 FIGURE 6.11; THE ROOSTING SITE OF AN ASIAN OPEN BILL STORK COLONY WITH NESTS
AND YOUNG CHICKS VISIBLE ....................................................................................................... 218 FIGURE 6.12; CLOSE CONTACT BETWEEN WILD BIRDS AND FARM DUCKS ............................. 219 FIGURE 7.1; PHYSICAL PATHWAY OF HPAI H5N1 TRANSMISSION ............................................... 226 FIGURE 7.2; RELEASE AND EXPOSURE PATHWAYS FOR RISK ASSESSMENTS
FIGURE 7.3; POTENTIAL PATHWAYS FOR SPREAD OF H5N1 BETWEEN BRIDGE SPECIES AND
OTHER AVIAN SPECIES FROM ONE AREA (A) TO ANOTHER AREA (B) ............................. 235 FIGURE 7.4; CUMULATIVE PROBABILITY DISTRIBUTION OF PREVALENCE OF LESSER
WHISTLING DUCKS ........................................................................................................................... 252 FIGURE 7.5; THE PROCESS OF CALCULATING P2 ............................................................................... 253 FIGURE 7.6; SENSITIVITY ANALYSIS OF THE P1XP2XP3XP4 MODEL* ......................................... 259 *TRUE PREVALENCE = P1, BETA/PASYM = PASYM, BETA/PSHED = PSHED .............................. 259 FIGURE 7.7; IMPACT OF THE SENSITIVITY OF THE WILD BIRD SURVEILLANCE PROGRAM
ON THE TRUE PREVALENCE OF AVIAN INFLUENZA H5N1 INFECTION IN LESSER
WHISTLING DUCKS ........................................................................................................................... 259 FIGURE 7.8; IMPACT OF THE SENSITIVITY OF THE WILD BIRD SURVEILLANCE PROGRAM
ON THE LESSER WHISTLING DUCKS’ OVERALL PROBABILITY ......................................... 260 FIGURE 7.9; AN OPEN SYSTEM DUCK FARM WITH A DUCK POND BETWEEN DUCK HOUSES
................................................................................................................................................................ 260 FIGURE 7.10; A BACKYARD CHICKEN, SIX WHITE VENTED MYNAS AND A PIGEON SHARING
THE SAME HABITAT ......................................................................................................................... 263 FIGURE 7.11; PIGEONS FEEDING IN A LAYER DUCK FARM ............................................................ 263