Thursday, December 20, 2012

Journal Atani Tokoyo: The identification of AI virus in ducks in Indonesia ckade 2.3.2

20 December 2012


The Eurasian lineage of H5N1 viruses continue to cause the highly pathogenic avian influenza (HPAI) in poultry in some countries in Asia and Africa. In Indonesia, H5N1 clade 2.1 viruses have been known to cause outbreaks of HPAI H5N1 in all roomates clade 2.1.3 viruses have predominantly Circulated in poultry in this country since 2005. Most H5N1 HPAI outbreak Occurs in chickens, whereas outbreaks in other avian species is rare Including ducks. However, between September and November 2012, several disease outbreaks were reported from duck farms in three provinces in Java (Central Java, Yogyakarta and East Java) with high morbidity and mortality seen in ducks. The majority of disease cases found in young ducks, but in some occasions, adult ducks were also affected. Neurological signs, whitish eye and death were the main clinical signs in young ducks, while reduced in egg production were frequently observed in affected laying ducks. Showed histopathology acute necrotic to chronic non-suppurative encephalitis and perivascular cuffing in dead or severe infected ducks. Immunohistochemistry result Showed H5N1 viral antigen detected mainly in brain. H5N1 virus was successfully isolated either from tissues, oropharyngeal and cloacal swabs or from feather samples. Avian influenza subtype H5 viral RNA was detected by real-time reverse transcription PCR. Phylogenetic analysis of hemagglutinin sequences of seven H5N1 virus isolates indicated that these isolates belong to clade 2.3.2, a sublineage that H5N1 has not been detected previously in Indonesia. Further analysis should be done to investigate Whether the Emergence of this virus in Indonesia is due to new H5N1 viral introduction or to mutation processes occurring in poultry. In addition, another study is Necessary to assess the pathogenecity of the virus in ducks and other poultry, Including chickens.


Disease highly pathogenic avian influenza (HPAI) caused by H5N1 subtype avian influenza virus was identified in poultry since 2003 (Dharmayanti et al ., 2004; Wiyono et al ., 2004). According to the classification of WHO / OIE / FAO, all H5N1 viruses isolated from poultry and humans in Indonesia, including the clade 2.1. The predominant H5N1 virus been found since 2005 to date comes from clade 2.1.3 (,, and Several studies have shown that infection with H5N1 virus-clade 2.1 in group chickens ( gallinaceous ) as layer chickens, broiler chicken, chicken is highly pathogenic, causing illness and death perakut high amounts, while ducks and other water fowl are relatively more resistant to infection these viruses ( Bingham et al. , 2009 ; Swayne, 2007 ; Wibawa et al. , 2012 ). The study results are consistent with the results of the investigation BBVet / BPPV and some sur-vei epidemiology and molecular epidemiology suggests that the rate of prevalence of H5N1 clade 2.1 in ducks and other water fowl in Indonesia is very low compared to the prevalence of the virus in chickens ( Henning et al. , 2010 ; Wibawa et al. , 2011 ; Loth et al. , 2011 ). In September-November 2012 reported the case of a fairly high mortality in ducks in Central Java, Yogyakarta and East Java DI. Center for Veterinary Wates (Wates BBVet) did invesitigasi in the field and conduct sampling in order to identify the causative agent of the death of the ducks. This paper aims to identify the causative agent of infectious diseases that are pathogenic to ducks. 


1. Disease Investigation Case

The investigation conducted by the activities of active disease in which the team BBVet Wates to respond actively, based on reports from farmers and agencies, to conduct on-site investigation of the case. In addition to servicing also active with passive submission service that evaluates agencies and individual samples (farmers), as well as the activities of semi-active / passive where samples were taken at Wates BBVet team doing vigorous activities on activity monitoring service or other animal disease surveillance. Investigation of cases of the disease in Central Java, Yogyakarta and East Java, conducted from September-November 2012 to follow up some duck deaths reported in several districts in the three provinces. Chronology of disease cases described in Table 1.
Description:     Mortality is calculated based on the number of deaths in the total population of ducks on a  farm, tc: there is no record as informal reports by farmers, td: not done.
Some information from the breeder also said that duck plague occurred in several districts in Central Java such Boyolali, Starch and Apex. Based on the results of investigations in the field and report deaths of ducks introductory sample data showed that the average mortality was 39.3% ducks with the lowest percentage of 8.3% and highest mortality reached 100.0%. 

2. Laboratory Testing

Laboratory testing conducted in BBVet Wates to know the main agent causing disease deaths in ducks. Some testing is done partly by surgical test carcasses, Rapid Test AI, histopathology, immunohistochemistry, virus isolation, conventionalpolymerase chain reaction (PCR) for the detection of ND virus, real-time reverse transcription PCR (RT-PCR) for the detection of influenza A virus ti-pe and subtype H5 viruses, bacterial culture and serologic testing (titer AI and ND).

 In addition to laboratory testing BBVet Wates, also tests DNA sequencing to determine the sequence of nucleotides (nucleic acids) that make up the gene hemagglutinin (HA) of influenza A virus from samples positive by the results of virus isolation and RT-PCR H5N1 subtype H5. From some of the positive samples, a total of 3-7 samples were sent to asequencing lab partner , the Center Veterinaria Farma (3 samples of duck), Central Veterinary Research Bogor (3 samples), Center for Veterinary Drug Testing and Certification Bogor (7 samples) and Hall Regional Veterinary Investigation II Bukittinggi (7 samples). Samples ducks 1-3, sent to all the sequencing lab partner, while No.. 4-7 sent to BBPMSOH and BPPV II Bukittinggi. Detailed penaamaan H5N1 virus isolates from ducks as below:
No. 1: A/duck/Sukoharjo/BBVW-1428-9/2012 
No. 2: A/duck/Bantul/BBVW-1443-9/2012 
No. 3: A/duck/Sleman/BBVW-1463-10/2012 
No. 4: A/duck/Wonogiri/BBVW-1730-11/2012 
No. 5: A/duck/Blitar/BBVW-1731-11/2012 
No. 6: A/duck/Tegal/BBVW-1727-11/2012 
No. 7: A / Muscovy duck/Tegal/BBVW-1732-11/2012

DNA sequencing was performed by the standard operation procedure (SOP) of the Australian Animal Health Laboratory(AAHL), Geelong Australia, using four pairs of specific primers were designed by the AAHL ( AAHL, 2008 ). Primers are designed to mensekuen whole HA gene fragment that da-pat obtained full open reading frame (ORF) of this gene ( AAHL, 2008 ).

3. Sequence analysis and phylogenetic

 DNASTAR Lasergene 8.0 software used for the assembly and editing HA gene sequences. Multiple alignment performed using the Clustal W in Bioedit software ( Hall, 1999 ). Construction of phylogenetic done in MEGA 4 software ( Tamura et al., 2007 ) with the method of Neighbour Joining (NJ) tree using 1000 bootstrap replication and Tamura-Nei93 (TN93) for models of nucleotide substitution. Analysis of nucleotides pair distances carried by p -distance replication Bootstrapping with 1000 models.


The results of laboratory testing

To find out the possible cause of duck plague inspection and testing has been carried out in the laboratory either by rapid test (Rapid Test) for AI, clinical examination, pathological anatomical, histopathological, immunohistochemical and serologic testing, bacteriology, virology and molecular biology with realtime RT- PCR using the primers and probes that identify specific AI subtypes H5. Test results virology and molecular biology in particular are presented in Table 2. 
On clinical examination of the diseased ducks, ducks seen that show clinical symptoms of nerve pain such as torticollis (Figure 1), tremors, difficulty standing, loss of balance when walking and in severe cases with death. Surgical outcomes carcasses not found specific changes except for a whitish cornea either unilateral or bilateral (Fig. 2), whitish streaks on the heart muscle that varies from mild to severe, and the congestion of the blood vessels and malasea (necrosis) of the brain with variations from mild to severe.
Histopathological examination showed infiltration of lymphocytes in high amounts in heart muscle. In multifocal acute brain inflammation and necrosis in the more chronic cases lymphocytes infiltrate the brain (Figure 3), followed by the inflammation of mild to severe perivascular cuffing. In immunohistochemical staining method using the antibody of H5N1 AI virus antigen is found in the cells of brain neurons (Fig. 4). 

Changes histopathologic and immunohistochemical results are similar to observations at the microscopic changes in experimental ducks were infected by H5N1 virus isolates from clade 1 or 2.1 ( Bingham et al. , 2009 ; Wibawa et al. , 2012 ).But the H5N1 viruses isolated from ducks cases recently seen having lesion severity higher than infections caused viruses from clade 2.1. Therefore, further research needs to be done to determine the extent pathogenesitas new isolates of different poultry species, especially chickens and ducks.

On examination of the bacterial culture of the eye, eye fluid, brain, heart and the hearts of all the results were negative fungal and bacterial pathogens. This suggests that the probability of death in ducks problem is not caused by bacteria and fungi infections. In serological tests, serum samples from 28 ducks found 8 samples tested (28.6%) AI H5 antibody positive and 12 samples (42.9%) positive antibody ND. With the antibody titers in both AI and ND duck serum, suggesting the possibility that ducks had been vaccinated or had the disease or ND AI field. From the test data found 1 virus isolation positive cases ND (samples from LDCC), AI 10 positive, 1 negative isolation and the remaining 5 cases are still in the process of virus isolation (Table 2). For ND virus isolates, the test was performed by injecting the corresponding virus isolates in ducks as intravenous way, however, after 3 weeks of ducks did not die and arise ND antibody with a high titer (HI titer 2 5 ). This result indicates that the ND virus isolates were found not to cause the death of duck plague. On examination by PCR, from 17 cases of ducks, 11 cases tested PCR and obtained the following data: 9 H5 viral RNA positive samples, 9 ND viral RNA negative, a negative sample either H5 or ND, and the rest are still in the process of testing (Table 2). This reinforces the results of molecular testing of suspected causes of death occurring outbreaks in ducks is AI virus subtypes H5.

Sequence and Phylogenetic Analysis of the hemagglutinin gene

The results showed that the DNA sequence of the ORF of the HA gene of the virus was 1707 base pairs ( base pairs ) and it encodes 569 amino acids of the HA protein. The seven isolates had high genetic similarity, which is 99% both at the level of genetic similarity nucleotides or amino acids. The results of the analysis of Basic Local Alignment Search Tools (BLAST) in Genbank and genetic distance using Mega 4 software ( Tamura et al. , 2007 ) showed that the seven isolates of H5N1 duck has a rate of 97-98% homology with H5N1 clade viruses Instead, based on, the level of homology with viruses from clade 2.1 low at around 91-93%. These results indicate that the H5N1 isolates from ducks is not derived from the Indonesian clade 2.1.

Sequence analysis showed that the HA protein isolates seven ducks have basic amino acid sequence motifs that recur tread area cuts protease enzyme ( proteolitic cleavage site ) were identical to viruses from clade, namely PQRERRRKR ( Li et al. , 2011 ) ( Figure 5). This indicates that the viruses isolated from ducks has Characteristic HPAI virus ( Perdue et al. , 1997 ; Senne et al. , 1996 ).

To view the classification of H5N1 isolates isolated from ducks, performed phylogenetic analyzes using the Neighbor-Joining (NJ) Tree with nekleotida TN93 substitution models using 1000 bootstrap replications. The results of the phylogenetic tree showed that the duck isolates included in clade 2.3.2 and is a branch of the phylogenetic clade (Figure 6). Next, to see whether this virus is still in one strain ( lineage ) with clade, the test was done to determine the genetic diversity within the average nucleotide pair duck isolates with viruses from clade WHO / OIE / FAO H5N1 Evolution Working Group ( WHO, 2008 ; WHO, 2012 ) has made ​​provision H5N1 clade classification as follows: 1) Classified a new clade if it has an average percentage range (diversity) nucleotide pair between species ( average pairwise distance ) of more than 1.5% of the clade that has been there and most previous definitions, 2) the results of phylogenetic analysis and the diversity of the HA sequence   shows sharing common ancestral node with bootstrap values> 60% of the nodes indicate phylogenetic clade (after 1000 neighbor-joining bootstrap replicates ).
Figure 6 . phylogenetic tree of H5N1 isolates isolated from ducks (3 isolates using that for analysis). Analysis using the NJ tree, with a nucleotide substitution model of Tamura-Nei (TN93) with 1000 boostrap replication. Phylogenetic tree dirootkan on A / goose / Guangdong/1/96 (H5N1). H5N1 isolates from ducks case is colored red.

Phylogenetic analysis and genetic diversity by using MEGA 4.0 software showed that the average distance between neighbor nucleotides pair of duck isolates was 0.3%, which means that the seven isolates of H5N1 from ducks case is still in the group, but the average distance of nucleotides pair with the group or other clusters within clade (group 1, group 2 and group 3) is more than 1.5% (2.3-4.6%) (Figure 6). Although the average distance between isolates nukletida pair of ducks is more than 1.5% of the other group in the clade, all virus isolates still share a common ancestral node of clade 2.3.2. These results indicate that there is a possibility isolates or ducks are derived from a group or cluster of new phylogenetic clade 2.3.2 (Fig. 6). To date it is known that only viruses derived clade of of 2.1 which attacks poultry and humans in Indonesia. With the invention of a new clade of H5N1 in Indonesia, particularly in the area of Yogyakarta, Central Java and East Java, suggesting the possibility of introduction of a new virus into Indonesia. But the initial introduction, the animal species involved, and the factors that cause the emergence of this virus which causes death in ducks in all three parts of Java is not yet known. To find it necessary to hold a retrospective study of both epidemiology and molecular epidemiology. More important is the need to be increased attention ( awareness ) and monitoring the possibility of expansion of the virus to other parts of Indonesia through the traffic of poultry or their products. To prevent the spread of cases, necessary control measures such depopuasi or culling of infected poultry ducks and restrictions and strict traffic control ducks and their products into and out of the three areas mentioned above.
  1. Based on the laboratory test results concluded that the suspected cause of duck plague that is currently happening in the province of Central Java, and East Java are DIYogyakarta AI subtype H5N1 disease.
  2. Seven isolates of H5N1 viruses have been sequenced allegedly not come from the lineage of clade 2.1 H5N1 virus has been endemic in poultry in Indonesia.
  3. Isolates of H5N1 viruses isolated from ducks have a higher degree of kinship to the viruses of clade (97-98% nucleic acid similarity) than kinship against viruses from clade 2.1 (91-93%).
  4. Based on phylogenetic analysis, isolates of H5N1 viruses isolated from ducks are included in clade 2.3.2.
  5. Based on the analysis of the genetic diversity of the nucleotide sequence of the HA gene, did not rule out if this virus isolates belong to a group ( sublineage ) new but still included in the clade 2.3.2. This should prove a more accurate analysis and involve more comprehensive virus isolates.
  6. The factors that led to the emergence of clade 2.3.2 into Indonesia needs to be further investigated, whether this is due to the introduction of a new virus into Indonesia.
  7. Need improved monitoring of poultry (chickens and waterfowl) on circulating viruses and the like clade 2.3.2 HPAI endemicity resulting.

The author would like to thank the Director of Animal Health, Head BBVet Wates, Chief Regional BPPV II Bukittingi, BBalitvet Chief, Head and Chief BBPMSOH Pusvetma for their input and support given in the writing of this article. Our thanks also go to the Australian Animal Health Laboratory (AAHL), Geelong, Australia and the FAO-OIE OFFLU Project, which has helped increase the capacity of the testing and development of AI and sequensing DNA diagnosis in laboratories in the technical services unit under the Directorate General of Livestock and Animal Health, Indonesia. We also would like to thank all parties, particularly the Department of Agriculture / Animal Husbandry and Animal Health at the district and also breeders who have helped in the investigation of disease.


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