Characteristics of two duck farming systems in the Mekong Delta of Viet Nam: stationary flocks and moving flocks, and their potential relevance to the spread of highly pathogenic avian influenza.

Ducks are considered to play a major role in the spread of highly pathogenic avian influenza (HPAI) H5N1 in Viet Nam, but detailed information on their management is limited. We distinguished two different systems (1) stationary duck flocks that are not commonly driven to rice fields beyond village boundaries and that are confined overnight on farms and (2) moving duck flocks that are intentionally driven to rice fields beyond village boundaries, that are not returning to home farms for extended periods and that are housed overnight in temporary enclosures in rice paddies. A total of 115 stationary and 22 moving flock farmers were interviewed in 2007 in the Mekong Delta of Viet Nam. Moving duck flocks are larger than stationary flocks, which is indicative of their more commercial production. Moving flock farmers apparently are more aware of HPAI risks than stationary flock farmers, as their flocks are more likely fully vaccinated and have less contact with chickens during scavenging. On the other hand, the spread of HPAI virus between birds might be promoted by moving duck flocks as they repeatedly use transport vehicles and numerous rice paddies for scavenging and are often visited by hatchery owners in the field for purchasing duck eggs. In addition, long distances travelled by moving duck flocks might also result in widespread dissemination of HPAI virus. Further studies are necessary to describe HPAI prevalence and travel patterns of moving duck flocks and to explore the moving duck flock network in detail.

Infection studies with two highly pathogenic avian influenza strains (Vietnamese and Indonesian) in Pekin ducks (Anas platyrhynchos), with particular reference to clinical disease, tissue tropism and viral shedding.

Pekin ducks were infected by the mucosal route (oral, nasal, ocular) with one of two strains of Eurasian lineage H5N1 highly pathogenic avian influenza virus: A/Muscovy duck/Vietnam/453/2004 and A/duck/Indramayu/BBVW/109/2006 (from Indonesia). Ducks were killed humanely on days 1, 2, 3, 5 and 7 after challenge, or whenever morbidity was severe enough to justify euthanasia. Morbidity was recorded by observation of clinical signs and cloacal temperatures; the disease was characterized by histopathology; tissue tropism was studied by immunohistochemistry and virus titration on tissue samples; and viral shedding patterns were determined by virus isolation and titration of oral and cloacal swabs. The Vietnamese strain caused severe morbidity with fever and depression; the Indonesian strain caused only transient fever. Both viruses had a predilection for a similar range of tissue types, but the quantity of tissue antigen and tissue virus titres were considerably higher with the Vietnamese strain. The Vietnamese strain caused severe myocarditis and skeletal myositis; both strains caused non-suppurative encephalitis and a range of other inflammatory reactions of varying severity. The principal epithelial tissue infected was that of the air sacs, but antigen was not abundant. Epithelium of the turbinates, trachea and bronchi had only rare infection with virus. Virus was shed from both the oral and cloacal routes; it was first detected 24 h after challenge and persisted until day 5 after challenge. The higher prevalence of virus from swabs from ducks infected with the Vietnamese strain indicates that this strain may be more adapted to ducks than the Indonesia strain.

A comparative evaluation of feathers, oropharyngeal swabs, and cloacal swabs for the detection of H5N1 highly pathogenic avian influenza virus infection in experimentally infected chickens and ducks.

Oropharyngeal and cloacal swabs have been widely used for the detection of H5N1 highly pathogenic avian Influenza A virus (HPAI virus) in birds. Previous studies have shown that the feather calamus is a site of H5N1 virus replication and therefore has potential for diagnosis of avian influenza. However, studies characterizing the value of feathers for this purpose are not available, to our knowledge; herein we present a study investigating feathers for detection of H5N1 virus. Ducks and chickens were experimentally infected with H5N1 HPAI virus belonging to 1 of 3 clades (Indonesian clades 2.1.1 and 2.1.3, Vietnamese clade 1). Different types of feathers and oropharyngeal and cloacal swab samples were compared by virus isolation. In chickens, virus was detected from all sample types: oral and cloacal swabs, and immature pectorosternal, flight, and tail feathers. During clinical disease, the viral titers were higher in feathers than swabs. In ducks, the proportion of virus-positive samples was variable depending on viral strain and time from challenge; cloacal swabs and mature pectorosternal feathers were clearly inferior to oral swabs and immature pectorosternal, tail, and flight feathers. In ducks infected with Indonesian strains, in which most birds did not develop clinical signs, all sampling methods gave intermittent positive results; 3-23% of immature pectorosternal feathers were positive during the acute infection period; oropharyngeal swabs had slightly higher positivity during early infection, while feathers performed better during late infection. Our results indicate that immature feathers are an alternative sample for the diagnosis of HPAI in chickens and ducks.

Efficacy of a high potency O1 Manisa monovalent vaccine against heterologous challenge with a FMDV O Mya98 lineage virus in pigs 4 and 7 days post vaccination.

Early protection with a high potency (>6PD50) foot-and-mouth disease (FMD) O1 Manisa (Middle-East South Asia lineage) vaccine against challenge with O/VIT/2010 (O Mya98 lineage) was tested in pigs. Only two pigs that were vaccinated seven days prior to challenge had any demonstrable antibodies as a result of vaccination at the time of challenge. However, 80% and 60% of pigs that were vaccinated seven and four days prior to coronary band challenge were protected. Vaccination significantly reduced the amount of virus excreted in nasal swabs, saliva and faeces compared to unvaccinated and infected controls. Virus and viral RNA could be detected in some pigs until termination of the experiment 14 days after challenge. Antibodies to the non-structural proteins (NSP) were detected in only one pig that was challenged four days post vaccination (dpv) and transiently in two pigs that were challenged sevendpv at only one time point. For each vaccine and control group, a group of unvaccinated pigs were kept in the same room but with no direct contact with the infected pigs to determine whether vaccination prevented transmission. Despite the presence of live virus and viral RNA in these indirect contact pigs, the groups in contact with the vaccinated and infected pigs did not develop clinical signs nor did they sero-convert. Contact pigs in the same room as unvaccinated challenged controls did show signs of disease and virus infection that resulted in sero-conversion to the NSP. A breach of the wall that separated the two groups at nine days post challenge might have contributed to this finding. This study showed that high potency vaccine can provide protection to pigs soon after vaccination and that aerosol transmission within rooms is a rare event.

A Malaysia 97 monovalent foot-and-mouth disease vaccine (>6PD50/dose) protects pigs against challenge with a variant FMDV A SEA-97 lineage virus, 4 and 7 days post vaccination.

Pigs play a significant role during outbreaks of foot-and-mouth disease (FMD) due to their ability to amplify the virus. It is therefore essential to determine what role vaccination could play to prevent clinical disease and lower virus excretion into the environment. In this study we investigated the efficacy of the double oil emulsion A Malaysia 97 vaccine (>6PD50/dose) against heterologous challenge with an isolate belonging to the A SEA-97 lineage at 4 and 7 days post vaccination (dpv). In addition, we determined whether physical separation of pigs in the same room could prevent virus transmission. Statistically there was no difference in the level of protection offered by 4 and 7 dpv. However, no clinical disease or viral RNA was detected in the blood of pigs challenged 4 dpv, although three of the pigs had antibodies to the non-structural proteins (NSPs), indicating viral replication. Viral RNA was also detected in nasal and saliva swabs, but on very few occasions. Two of the pigs vaccinated seven days prior to challenge had vesicles distal from the injection site, but on the inoculated foot, and two pigs had viral RNA detected in the blood. One pig sero-converted to the NSPs. In contrast, all unvaccinated and inoculated pigs had evidence of infection. No infection occurred in any of the susceptible pigs in the same room, but separated from the infected pigs, indicating that strict biosecurity measures were sufficient under these experimental conditions to prevent virus transmission. However, viral RNA was detected in the nasal swabs of one group of pigs, but apparently not at sufficient levels to cause clinical disease. Vaccination led to a significant decrease in viral RNA in vaccinated pigs compared to unvaccinated and infected pigs, even with this heterologous challenge, and could therefore be considered as a control option during outbreaks.

Farm- and flock-level risk factors associated with Highly Pathogenic Avian Influenza outbreaks on small holder duck and chicken farms in the Mekong Delta of Viet Nam.

After 11 consecutive months of control, the Mekong Delta in Viet Nam experienced a wave of Highly Pathogenic Avian Influenza (HPAI) H5N1 outbreaks on small holder poultry farms from December 2006 to January 2007. We conducted a retrospective matched case-control study to investigate farm- and flock-level risk factors for outbreak occurrence during this period. Twenty-two case farms were selected from those where clinical signs consistent with HPAI H5N1 had been present and HPAI H5N1 had been confirmed with a positive real-time PCR test from samples obtained from affected birds. For every case farm enrolled, two control farms were selected matched on time of outbreak occurrence, farm location and species. Veterinarians conducted interviews with farmers, to collect information on household demographics, farm characteristics, husbandry practices, trading practices, poultry health, vaccination and biosecurity. Exact stratified logistic regression models were used to assess putative risk factors associated with a flock having or not having a HPAI outbreak. Nested analyses were also performed, restricted to subsets of farms using scavenging, confinement or supplementary feeding practices. Risk of an outbreak of HPAI H5N1 was increased in flocks that had received no vaccination (odds ratio (OR)=20.2; 95% confidence interval (CI): 1.0, +infinity) or only one vaccination (OR=85.2; 95% CI: 6.5, +infinity) of flocks compared to two vaccinations, and in flocks on farms that had family and friends visiting (OR=8.2; 95% CI: 1.0, +infinity) and geese present (OR=11.5; 95% CI: 1.1, +infinity). The subset analysis using only flocks that scavenged showed that sharing of scavenging areas with flocks from other farms was associated with increased risk of an outbreak (OR=10.9; 95% CI: 1.4, 492.9). We conclude that none or only one vaccination, visitors to farms, the presence of geese on farms and sharing of scavenging areas with ducks from other farms increase the risk of HPAI H5N1 outbreaks in poultry flocks in Viet Nam.

Yemen and Vietnam capripoxviruses demonstrate a distinct host preference for goats compared with sheep.

Sheeppox and goatpox are caused by viruses that are members of the genus Capripoxvirus, and globally result in significant production losses. To improve the understanding of disease pathogenesis and evaluate host species preferences, sheep and goats were inoculated either with a capripoxvirus isolate from Yemen or from a recent outbreak in Vietnam. Blood, swabs and tissues were collected at various time points following experimental challenge and assessed for viral DNA content using real-time PCR and infectivity using virus isolation. The Yemen isolate was considerably more pathogenic in goats with 100 % mortality and morbidity compared with sheep with 0 % mortality and 100 % morbidity. The Vietnam isolate was also more pathogenic in goats with 100 % morbidity and an estimated 33 % mortality rate compared with mild morbidity and a 0 % mortality rate in sheep. Higher viral titres were observed in nasal, oral and conjunctival swabs from goats inoculated with either the Yemen or Vietnam isolate compared with those collected from sheep. Although the highest viral titres were detected in primary and secondary skin lesions in sheep and goats, the severity of clinical disease observed in each species varied according to the inoculum used. Whereas both the Yemen and Vietnam isolates clearly caused more severe disease in goats, the Yemen isolate was also moderately pathogenic in sheep. The Vietnam isolate, in contrast, caused only very mild disease in sheep. Limited DNA sequencing revealed ORF 074 of the Vietnam isolate to be identical to that of several goatpox virus isolates from China, suggesting a possible Chinese origin.