Risk mapping using serologic surveillance for selected One Health and transboundary diseases in Cambodian goats.

In Cambodia, goat production and meat consumption are customary among Muslim communities. Recently, goat meat has gained popularity among Cambodians. Goat farmers use a traditional management system, including grazing, requiring minimal labour. The close proximity between humans and animals could increase the risk of zoonotic disease transmission. A serological survey was undertaken to estimate the prevalence of some priority zoonoses and high-impact animal diseases in the Cambodian goat population. A total of 540 samples were collected from goats in six provinces and analysed with commercially available enzyme-linked immunosorbent assays for Brucella species, Q fever (Coxiella burnetii), Foot and Mouth Disease virus non-structural protein (FMDV NSP) and Peste des Petits Ruminants virus (PPRV). True seroprevalences with a 95% Confidence Interval (CI), taking into account imperfect tests, risk factors and odds ratios (ORs), were calculated to better understand the disease distribution and epidemiology. Independent variables used in statistical modellings included sex, body condition score, age, vaccination history, province and commune, while dependent variables were ELISA test results. The overall true prevalence of antibodies to Brucella spp., C. burnetii, FMDV and PPRV, were 0.1% (95% CI 0.0, 1.0), 7.2% (95% CI 5.3, 9.7), 57.7% (95% CI 53.1, 62.3) and 0.0% (95% CI 0.0, 0.0), respectively. There was no identified risk factor for brucellosis and PPR. The two risk factors for C. burnetii seropositivity were sex (p-value = 0.0005) and commune (p-value <0.0001). However, only the OR of C. burnetii seropositive female goat was significant at 9.7 (95% CI 2.7, 35.5) times higher than male. The risk factors of FMD NSP seropositivity were age (p-value = 0.001) and commune (p-value <0.0001). Only the age 'more than two-year-old' group with a significant OR of 6.2 (95% CI 2.1, 18.4) using the 'up to one-year-old' group as the reference. In summary, Brucella spp. seroprevalence was low, while no evidence of PPRV antibodies was detected in the goat populations. C. burnetii seroprevalence in female goats was significantly higher than for males, and there were significant differences in C. burnetii seroprevalence between communes. The overall FMDV NSP seroprevalence was high, especially in older animals. Vaccination should be advocated to protect animals from FMDV and improve productivity. As the impacts of these zoonoses on human and animal health were still unknown, further investigation of these zoonotic diseases' epidemiology is recommended.

Utilising abattoir sero-surveillance for high-impact and zoonotic pig diseases in Lao PDR.

National disease surveillance systems are essential to a healthy pig industry but can be costly and logistically complex. In 2019, Lao People's Democratic Republic (Lao PDR) piloted an abattoir disease surveillance system to assess for the presence of high impact pig diseases (HIPDs) using serological methods. The Lao Department of Livestock and Fisheries (DLF) identified Classical Swine Fever (CSF), Porcine Respiratory and Reproductive Syndrome (PRRS) and Brucella suis as HIPDs of interest for sero-surveillance purposes. Porcine serum samples (n = 597) were collected from six Lao abattoirs in March to December of 2019. Serological enzyme-linked immunosorbent assay (ELISA) methods were chosen for their high-throughput and relatively low-costs. The true seroprevalence for CSF and PRRS seropositivity were 68.7%, 95% CI (64.8-72.3) and 39.5%, 95% CI (35.7-43.5), respectively. The results demonstrated no evidence of Brucella spp. seroconversion. Lao breed pigs were less likely to be CSF seropositive (P < 0.05), whilst pigs slaughtered at <1 year of age were less likely to be PRRS seropositive (P < 0.01). The testing methods could not differentiate between seropositivity gained from vaccine or natural infection, and investigators were unable to obtain the vaccine status of the slaughtered pigs from the abattoirs. These results demonstrate that adequate sample sizes are possible from abattoir sero-surveillance and lifetime health traceability is necessary to understand HIPDs in Lao PDR.

Abattoir-based serological surveillance for transboundary and zoonotic diseases in cattle and swine in Cambodia: a pilot study in Phnom Penh province during 2019 and 2020.

A pilot animal disease surveillance program was implemented at four abattoirs in Phnom Penh, Cambodia, between October 2019 and January 2020. A total of 1141 samples were collected from 477 cattle and 664 swine. Serological testing was performed using commercial antibody ELISA kits for zoonotic and high-impact animal diseases, namely brucellosis, Q fever, classical swine fever (CSF), porcine reproductive and respiratory syndrome (PRRS) and African swine fever (ASF). Only two samples tested positive for Brucella antibodies (0.2%, 95% CI 0.4-0.6, n = 1141). The seroprevalence of Q fever was 0.8% (95% CI 0.3-2.1, n = 477) in the cattle samples, while CSF, PRRS and ASF in pigs were 55.4% (95% CI 51.6-59.2, n = 655), 81.2% (95% CI 78.1-84.0, n = 655) and 2.6% (95% CI 1.6-4.1, n = 664), respectively. All 38 doubtful and 17 positive ASF antibody ELISA samples were negative when tested by real-time PCR. Univariate analyses demonstrated that the factor significantly associated with positive results of ASF was the abattoir location (p-value = 0.002). Based on logistic regression models, significant risk factors for CSF were province of origin (p-value = 1.7 × 10-6), abattoir (p-value = 3.6 × 10-11) and PRRS positivity (p-value = 0.004), and for PRRS were province of origin (p-value = 0.0004) and CSF positivity (p-value = 0.001). In conclusion, the seroprevalences of zoonotic diseases in this study were very low. The high prevalence of CSF and PRRS antibodies were most likely the result of vaccination. All ASF seropositive pigs, including those that gave equivocal results, originated from large-scale Cambodian-based commercial farms, as well as Thailand, which raises questions about possible illegal vaccination or low-pathogenicity ASF variants. The pilot abattoir serological surveillance program described here has the potential to provide a sentinel for incursions of novel and endemic pathogens, although further work is required to demonstrate its capacity to provide information on the longitudinal disease trends.

Abattoir-Based Serological Surveillance and Spatial Risk Analysis of Foot-and-Mouth Disease, Brucellosis, and Q Fever in Lao PDR Large Ruminants.

A national animal disease surveillance network initiated by the Lao PDR government is adopted and reinforced by a joint research project between the National Animal Health Laboratory (NAHL), the Department of Livestock and Fisheries (DLF), and the Mahidol Oxford Tropical Medicine Research Unit (MORU). The network is strengthened by staff training and practical exercises and is utilised to provide zoonotic or high-impact disease information on a national scale. Between January and December 2020, large ruminant samples are collected monthly from 18 abattoirs, one in each province, by provincial and district agriculture and forestry officers. The surveillance network collected a total of 4247 serum samples (1316 buffaloes and 2931 cattle) over this period. Samples are tested for antibodies against Brucella spp., Coxiella burnetii (Q fever) and Foot-and-Mouth Disease Non-Structural Protein (FMD NSP) using commercial ELISA kits and the Rose Bengal test. Seroprevalences of Q fever and brucellosis in large ruminants are low at 1.7% (95% CI: 1.3, 2.1) and 0.7% (95% CI: 0.5, 1.0) respectively, while for FMD NSP it is 50.5% (95% CI: 49.0, 52.0). Univariate analyses show differences in seroprevalences of Q fever between destination (abattoir) province (p-value = 0.005), province of origin (p-value = 0.005), animal type (buffalo or cattle) (p-value = 0.0008), and collection month (p-value = 3.4 × 10−6). Similar to Q fever, seroprevalences of brucellosis were significantly different for destination province (p-value < 0.00001), province of origin (p-value < 0.00001), animal type (p-value = 9.9 × 10−5) and collection month (p-value < 0.00001), plus body condition score (p-value = 0.003), and age (p-value = 0.007). Additionally, risk factors of the FMD NSP dataset include the destination province (p-value < 0.00001), province of origin (p-value < 0.00001), sex (p-value = 7.97 × 10−8), age (p-value = 0.009), collection date (p-value < 0.00001), and collection month (p-value < 0.00001). Spatial analyses revealed that there is no spatial correlation of FMD NSP seropositive animals. High-risk areas for Q fever and brucellosis are identified by spatial analyses. Further investigation of the higher risk areas would provide a better epidemiological understanding of both diseases in Lao PDR. In conclusion, the abattoir serological survey provides useful information about disease exposure and potential risk factors. The network is a good base for field and laboratory staff training in practical technical skills. However, the sustainability of such a surveillance activity is relatively low without an external source of funding, given the operational costs and insufficient government budget. The cost-effectiveness of the abattoir survey could be increased by targeting hotspot areas, reducing fixed costs, and extending the focus to cover more diseases.

Seroepidemiology of Foot and Mouth Disease using passive surveillance techniques in selected provinces of Lao PDR.

Foot and Mouth Disease (FMD) is a high-impact, contagious transboundary animal disease that is endemic in Southeast Asia. Abattoir samples were routinely collected in six selected provinces between March and December 2019. A total of 1280 samples of abattoir animals were tested for FMD Non-Structural Protein (NSP) antibodies to indicate natural infections. Overall, 22.8% were seropositive for FMD NSP antibodies while seroprevalence of cattle (n = 469), buffalo (n = 214), and pigs (n = 597) were 44.6%, 35.0%, and 1.3%, respectively. The highest seroprevalence destination province was Xiengkhouang (35.3% of 272 samples), followed by Savannakhet (27.0% of 244 samples). Risk factors for evidence of natural infection identified by a multivariate logistic regression model included age groups (p-value = 0.02) and origin provinces (p-value = 2.8 × 10-5) of the animals. There were significant differences of FMD NSP seroprevalence between age groups and origin provinces of the animals. The odds ratio of a seropositive result in the less than 1 year old group was 2.5 (95% CI; 1.4, 4.4) when compared to the 3-4 years old group, while the odds ratios for animals that originated from Khammouane and Xiengkhouang provinces were 4.5 (95% CI; 1.1, 18.7) and 2.4 (95% CI; 1.4, 4.1), respectively, when compared to Champasak province. Serotype-specific antibody ELISA for 44 NSP antibody-positive samples revealed evidence of FMD serotypes O and A virus circulation in some provinces. Despite the passive abattoir survey providing useful information on FMD virus previous exposure and geographic locations of the animals, timely information on FMD virus circulation and distribution is also crucial to an effective control program. Alternative approaches to increase the cost-effectiveness of the surveillance network are also discussed.

The Development of an Abattoir-Based Surveillance System in Lao PDR for the Detection of Zoonoses in Large Ruminants: Q Fever and Brucellosis Seroepidemiology as a Pilot Study.

Although animal health surveillance programmes are useful for gaining information to help improve global health and food security, these programmes can be challenging to establish in developing economies with a low-resource base. This study focused on establishing a national surveillance system initiated by the Lao PDR government using a passive surveillance system of abattoir samples as a pilot model, and to gain information on contagious zoonoses, particularly Q fever and brucellosis, in the large ruminant population. A total of 683 cattle and buffalo samples were collected from six selected provinces of Lao PDR between March-December 2019. Out of 271 samples tested, six samples (2.2%, 95% confidence interval (CI) of 1.0, 4.8) were positive in the Q fever antibody ELISA test. Only one sample (out of 683; 0.2%, 95% CI 0.0, 0.8) tested positive to the Brucella antibody ELISA test. Seroprevalence of these important zoonoses in Lao PDR were relatively low in cattle and buffaloes; however, extensive animal movement within the country was identified which could increase risks of spreading transboundary diseases. The study highlights the importance of ongoing animal health surveillance and the need to find cost-effective approaches for its long-term sustainability.

Serosurveillance of Coxiellosis (Q-fever) and Brucellosis in goats in selected provinces of Lao People's Democratic Republic.

Goat raising is a growing industry in Lao People's Democratic Republic, with minimal disease investigation to date, especially zoonoses. This study determined the proportional seropositivity of two zoonotic diseases: Q fever (causative agent Coxiella burnetii) and Brucellosis (Brucella species) in goats across five provinces (Vientiane Capital, Xayaboury, Xiengkhuang, Savannakhet and Attapeu). A total of 1458 goat serum samples were tested using commercial indirect ELISA for both pathogens, plus Rose Bengal agglutination test for Brucellosis. Overall individual seropositivity of C. burnetii was 4.1% and Brucella spp. was 1.4%. A multiple logistic regression model identified that province (Vientiane Capital, p = 0.05), breed (introduced Boer mixed breed, p = 0.006) and age (goats ≥3 years old, p = 0.014) were significant risk factors for C. burnetii seropositivity. The results of the survey indicated that province (Vientiane Capital, p<0.001), breed (introduced Boer mixed breed, p<0.001), production system (commercial, p<0.001), age (adult, p = 0.004), and farm size (large, 0.001) were all significant risk factors seropositivity for Brucella spp. It was concluded that Lao goats have been exposed to both C. burnetii and Brucella spp. however the risk of clinical disease has not yet been determined and there is an urgent need to determine human health risks and economic losses caused by Q fever and Brucellosis.

Identification of Indonesian clade 2.1 highly pathogenic influenza A(H5N1) viruses with N294S and S246N neuraminidase substitutions which further reduce oseltamivir susceptibility.

We have tested the in vitro susceptibility to the neuraminidase (NA) inhibitors of 96 highly pathogenic clade 2.1 A(H5N1) viruses from Indonesia, isolated between 2008 and 2011. HPAI virus samples obtained through the Influenza Virus Monitoring (IVM) surveillance program in Indonesia were tested for susceptibility to oseltamivir and zanamivir. The NAs of four viruses were identified as extreme outliers to oseltamivir, based on statistical analysis by box plots, with IC50 values ranging from 46 to 62 nM. The NAs of two of these viruses from Sumatra and Aceh, had an N294S substitution, while one virus from Sulawesi had an S246N NA substitution. The NAs of all four viruses showed a specific loss of slow binding to oseltamivir in an IC50 kinetics assay. As observed in our previous surveillance, there was only a minimal effect on the sensitivity to zanamivir or peramivir for these mutants or any of the other isolates tested. The continued circulation of subtype H5N1 viruses in avian species poses an on-going zoonotic threat. The fact that we continue to identify avian isolates with naturally occurring mutations conferring reduced oseltamivir susceptibility remains a concern, given oseltamivir will be a key antiviral in the event of a new pandemic emerging.

Developing Farm-Level Post-vaccination Sero-Monitoring Systems for H5N1 Highly Pathogenic Avian Influenza in an Endemically Infected Country.

Whilst the serological responses of poultry following vaccination against highly pathogenic avian influenza H5N1 has been extensively investigated under laboratory conditions, there have been fewer studies conducted in the field. This applies particularly to the endemically infected countries routinely practicing vaccination, where the combination of multiple circulating clades and/or the use of vaccines with different seed strains makes the design and interpretation of field studies especially problematic. To address this for the particular situation of layer hens in the small to medium commercial sector in Indonesia, we developed a sampling regime before and after the vaccination given to point-of-lay pullets, and assessed serological response with a panel of test antigens. This confirmed that high titres were induced in those birds vaccinated with locally produced homologous H5N1 vaccines administered two or more times, but in flocks using imported heterologous H5N2 vaccines median titres were significantly lower, and unlikely to provide protection throughout the production cycle, without additional vaccination. Comparing the HI responses against the panel of antigens enabled the detection of the flock's exposure to different vaccine antigens, and made possible the detection of mislabelled vaccine seed strains. Furthermore, we show that test antigens need not be exactly matched to assess sero-protection in well vaccinated birds. Finally our study suggests that the POL vaccination serves as a useful reference point for following cohorts of layers throughout their production cycle, and thus enabling robust vaccination field effectiveness studies.

Seroprevalence of Q Fever, Brucellosis, and Bluetongue in Selected Provinces in Lao People's Democratic Republic.

This study has determined the proportional seropositivity of two zoonotic diseases, Q fever and brucellosis, and bluetongue virus (BTV) which is nonzoonotic, in five provinces of Lao People's Democratic Republic (PDR) (Loungphabang, Luangnumtha, Xayaboury, Xiengkhouang, and Champasak, and Vientiane Province and Vientiane capital). A total of 1,089 samples from buffalo, cattle, pigs, and goats were tested, with seropositivity of BTV (96.7%), Q fever (1.2%), and brucellosis (0.3%). The results of this survey indicated that Q fever seropositivity is not widely distributed in Lao PDR; however, Xayaboury Province had a cluster of seropositive cattle in seven villages in four districts (Botan, Kenthao, Paklaiy, and Phiang) that share a border with Thailand. Further studies are required to determine if Xayaboury Province is indeed an epidemiological hot spot of Q fever activity. There is an urgent need to determine the levels of economic loss and human health-related issues caused by Q fever, brucellosis, and BTV in Lao PDR.

Evaluation of a conserved HA274-288 epitope to detect antibodies to highly pathogenic avian influenza virus H5N1 in Indonesian commercial poultry.

A peptide enzyme linked immunosorbent assay (ELISA) based on an epitope in the haemagglutinin (HA) of avian influenza virus H5N1, amino acid positions 274-288 (HA274-288) was evaluated for detection of H5N1-specific antibodies. An optimized ELISA based on the tetrameric form of the HA274-288 epitope designated MP15 gave low background with non-immune chicken sera and detected vaccinated and infected birds. The HA274-288 epitope was highly conserved in Indonesian H5N1 strains and antibody responses were detected in the majority of the vaccinated chickens regardless of the H5N1 strain used for vaccination. The HA274-288 epitope was also conserved in the majority of H5N1 strains from the neighbouring Asian region, and other H5 subtypes potentially allowing for a wider use of the MP15 ELISA in H5N1 vaccinated and infected flocks. The MP15 ELISA results correlated significantly with haemagglutination inhibition (HI) test results and test sensitivity and specificity were 87% and 92%, respectively. The MP15 ELISA titres were significantly higher than the HI titres in all immune sera allowing for sera to be tested at a single dilution of 1:400 which is of advantage in routine surveillance. The study indicated that the MP15 ELISA is potentially useful for serological detection of H5N1 vaccinated or infected poultry and to have some advantages over the standard HI test for routine monitoring of flocks' immunity after vaccination.

Surveillance at the molecular level: Developing an integrated network for detecting variation in avian influenza viruses in Indonesia.

Since 2006, Indonesia has used vaccination as the principal means of control of H5N1-HPAI. During this time, the virus has undergone gradual antigenic drift, which has necessitated changes in seed strains for vaccine production and associated modifications to diagnostic antigens. In order to improve the system of monitoring such viral evolution, the Government of Indonesia, with the assistance of FAO/OFFLU, has developed an innovative network whereby H5N1 isolates are antigenically and genetically characterised. This molecular surveillance network ("Influenza Virus Monitoring" or "IVM") is based on the regional network of veterinary diagnostic laboratories, and is supported by a web-based data management system ("IVM Online"). The example of the Indonesian IVM network has relevance for other countries seeking to establish laboratory networks for the molecular surveillance of avian influenza and other pathogens.

Hendra virus survival does not explain spillover patterns and implicates relatively direct transmission routes from flying foxes to horses.

Hendra virus (HeV) is lethal to humans and horses, and little is known about its epidemiology. Biosecurity restrictions impede advances, particularly on understanding pathways of transmission. Quantifying the environmental survival of HeV can be used for making decisions and to infer transmission pathways. We estimated HeV survival with a Weibull distribution and calculated parameters from data generated in laboratory experiments. HeV survival rates based on air temperatures 24 h after excretion ranged from 2 to 10 % in summer and from 12 to 33 % in winter. Simulated survival across the distribution of the black flying fox (Pteropus alecto), a key reservoir host, did not predict spillover events. Based on our analyses we concluded that the most likely pathways of transmission did not require long periods of virus survival and were likely to involve relatively direct contact with flying fox excreta shortly after excretion.

Antibody titer has positive predictive value for vaccine protection against challenge with natural antigenic-drift variants of H5N1 high-pathogenicity avian influenza viruses from Indonesia.

UNLABELLED: Vaccines are used in integrated control strategies to protect poultry against H5N1 high-pathogenicity avian influenza (HPAI). H5N1 HPAI was first reported in Indonesia in 2003, and vaccination was initiated in 2004, but reports of vaccine failures began to emerge in mid-2005. This study investigated the role of Indonesian licensed vaccines, specific vaccine seed strains, and emerging variant field viruses as causes of vaccine failures. Eleven of 14 licensed vaccines contained the manufacturer's listed vaccine seed strains, but 3 vaccines contained a seed strain different from that listed on the label. Vaccines containing A/turkey/Wisconsin/1968 (WI/68), A/chicken/Mexico/28159-232/1994 (Mex/94), and A/turkey/England/N28/1973 seed strains had high serological potency in chickens (geometric mean hemagglutination inhibition [HI] titers, ≥ 1:169), but vaccines containing strain A/chicken/Guangdong/1/1996 generated by reverse genetics (rg; rgGD/96), A/chicken/Legok/2003 (Legok/03), A/chicken/Vietnam/C57/2004 generated by rg (rgVN/04), or A/chicken/Legok/2003 generated by rg (rgLegok/03) had lower serological potency (geometric mean HI titers, ≤ 1:95). In challenge studies, chickens immunized with any of the H5 avian influenza vaccines were protected against A/chicken/West Java/SMI-HAMD/2006 (SMI-HAMD/06) and were partially protected against A/chicken/Papua/TA5/2006 (Papua/06) but were not protected against A/chicken/West Java/PWT-WIJ/2006 (PWT/06). Experimental inactivated vaccines made with PWT/06 HPAI virus or rg-generated PWT/06 low-pathogenicity avian influenza (LPAI) virus seed strains protected chickens from lethal challenge, as did a combination of a commercially available live fowl poxvirus vaccine expressing the H5 influenza virus gene and inactivated Legok/03 vaccine. These studies indicate that antigenic variants did emerge in Indonesia following widespread H5 avian influenza vaccine usage, and efficacious inactivated vaccines can be developed using antigenic variant wild-type viruses or rg-generated LPAI virus seed strains containing the hemagglutinin and neuraminidase genes of wild-type viruses. IMPORTANCE: H5N1 high-pathogenicity avian influenza (HPAI) virus has become endemic in Indonesian poultry, and such poultry are the source of virus for birds and mammals, including humans. Vaccination has become a part of the poultry control strategy, but vaccine failures have occurred in the field. This study identified possible causes of vaccine failure, which included the use of an unlicensed virus seed strain and induction of low levels of protective antibody because of an insufficient quantity of vaccine antigen. However, the most important cause of vaccine failure was the appearance of drift variant field viruses that partially or completely overcame commercial vaccine-induced immunity. Furthermore, experimental vaccines using inactivated wild-type virus or reverse genetics-generated vaccines containing the hemagglutinin and neuraminidase genes of wild-type drift variant field viruses were protective. These studies indicate the need for surveillance to identify drift variant viruses in the field and update licensed vaccines when such variants appear.

Novel phlebovirus with zoonotic potential isolated from ticks, Australia.

Recently discovered tick-borne phleboviruses have been associated with severe disease and death among persons in Asia and the United States. We report the discovery of a novel tick phlebovirus in Tasmania State, Australia, that is closely related to those zoonotic viruses found in Asia and North America.

Evaluation of avian influenza serologic and virologic diagnostic methods in wild Anseriformes and Charadriiformes.

Evaluation of avian influenza virus (AIV) diagnostic methods, including a nucleoprotein (NP) competitive enzyme-linked immunosorbent assay (c-ELISA), hemagglutination inhibition (HI) test, type A real-time reverse transcription polymerase chain reaction (RRT-PCR), and embryonating chicken egg (ECE) virus isolation (VI), suggested validity of these tests in wild birds comparable to that reported in poultry. This was determined by analyzing the results from experimental inoculation of three species of wild birds with a low-pathogenicity AIV and from field surveillance data. The NP c-ELISA in a high-AIV prevalence setting had 100% diagnostic sensitivity (Se; 95% confidence interval [CI]: 81.5%-100%) and 91% diagnostic specificity (Sp; 95% CI: 70.8%-98.9%) in negative controls compared with the RRT-PCR. In low-AIV prevalence flocks using a > 60% inhibition positivity threshold, relative to the HI test, c-ELISA performed with 90.5% Se (95% CI: 86.2%-93.8%) and 41.2% Sp (95% CI: 38.1%-44.5%). Assessment of HI suggests a titer > or = 8 is a positive test result in wild-bird sera, and using this titer had 83.3% Se (95% CI: 58.6%-96.4%) in experimentally infected birds. The RRT-PCR diagnostic performance compared with VI in cloacal swabs varied over 2-6 days postinoculation, having high Se (83.3%-100%) and Sp (94.1%-100%) with substantial agreement (kappa = 0.8). The cycle thresholds (C(t)) for the RRT-PCR of C(t) < 37 for positivity and C(t) = 37-40 as indeterminate were found to be valid for the species included in this study. In view of the interpretative diagnostic difficulties in heterogeneous populations of wild birds, this evaluation in three species of wild birds and in surveillance data should provide greater confidence in the application of these methods routinely used in poultry.

H5N1 infection causes rapid mortality and high cytokine levels in chickens compared to ducks.

Infection with H5N1 influenza virus is often fatal to poultry with death occurring in hours rather than days. However, whilst chickens may be acutely susceptible, ducks appear to be asymptomatic to H5N1. The mechanisms of disease pathogenesis are not well understood and the variation between different species requires investigation to help explain these species differences. Here we investigated the expression of several key proinflammatory cytokines of chickens and ducks following infection with 2 highly pathogenic H5N1 (A/Muscovy duck/Vietnam/453/2004 (Vt453) and A/Duck/Indramayu/BBVW/109/2006 (Ind109)) and a low-pathogenic H5N3 influenza virus (A/Duck/Victoria/1462/2008 (Vc1462)). H5N1 viruses caused fatal infections in chickens as well as high viral loads and increased production of proinflammatory molecules when compared to ducks. Cytokines, including Interleukin 6 (IL6) and the acute phase protein Serum Amyloid A (SAA), were rapidly induced at 24h post infection with H5N1. In contrast, low induction of these cytokines appeared in ducks and only at later times during the infection period. These observations support that hypercytokinemia may contribute to pathogenesis in chickens, whilst the lower cytokine response in ducks may be a factor in their apparent resistance to disease and decreased mortality.

Development of veterinary laboratory networks for avian influenza and other emerging infectious disease control: the southeast asian experience.

The outbreak of highly pathogenic H5N1 avian influenza, with its international spread, confirmed that emerging infectious disease control must be underpinned by effective laboratory services. Laboratory results are the essential data underpinning effective surveillance, case diagnosis, or monitoring of responses. Importantly, laboratories are best managed within national and international networks of technological support rather than in isolation. A well planned laboratory network can deliver both a geographical spread of testing capacity and also a cost effective hierarchy of capability. Hence in the international context regional networks can be particularly effective. Laboratories are an integral part of a country's veterinary services and their role and function should be clearly defined in the national animal health strategy and supporting government policies. Not every laboratory should be expected to deliver every possible service, and integration into regional and broader international networks should be a part of the overall strategy. The outputs required of each laboratory should be defined and then ensured through accredited quality assurance. The political and scientific environment in which laboratories operate changes continuously, not only through evolving national and regional animal health priorities but also through new test technologies and enhancements to existing technologies. Active networks help individual laboratories to monitor, evaluate, and respond to such challenges and opportunities. The end result is enhanced emerging infectious disease preparedness across the region.

Variation in the responses of wild species of duck, gull, and wader to inoculation with a wild-bird-origin H6N2 low pathogenicity avian influenza virus.

There is poor understanding of host responses to avian influenza virus (AIV) infection in wild birds, with most experimental studies using captive-bred birds and highly pathogenic AIVs that have an early endpoint. The objective of this study was to experimentally assess antibody responses and patterns of viral excretion in wild birds challenged with a low pathogenicity AIV. Ruddy turnstones (Arenaria interpres), silver gulls (Chroicocephalus novaehollandiae), and wandering whistling ducks (Dendrocygna arcuata) were challenged with a H6N2 virus, and blood, cloacal, and oropharyngeal (OP) swabs were analyzed from each bird over 28 days, with serology conducted on the ducks for a further 7 mo. Nineteen of 22 birds showed evidence of infection, with respiratory infection prevalent in the turnstones and gulls as mostly low titer viral excretion to 4 days postinoculation (DPI) with gastrointestinal replication detected in only one turnstone. In AIV naive ducks, there was gastrointestinal tropism with moderately high titer viral excretion via the cloaca to 6 DPI and low-grade OP viral excretion to 4 DPI. The hemagglutination inhibition antibody response was poor in the ducks, declining from 19 to 56 DPI, with higher titer responses in the gulls and turnstones. All infected birds responded with elevated nucleoprotein antibodies (in competitive enzyme-linked immunosorbent assay) by 7-10 DPI, and in the ducks these waned slowly after 42 DPI and were long-lived to at least 8 mo. The interspecies variability in response was consistent with a subtype that had adapted well in ducks, while the response of the turnstones may have been influenced by preexisting immunity to AIV. These findings provide insight into AIV infection dynamics in wild birds and highlight the need for further research.

Molecular epidemiology of influenza A (H5N1) viruses, Bangladesh, 2007-2011.

To investigate the origins, evolution and patterns of spread of HPAI H5N1 outbreaks in Bangladesh, we performed a phylogenetic reconstruction analysis using Bayesian methods. The analysis was conducted using 81 hemagglutinin (HA) gene sequences from the H5N1 viruses isolated in Bangladesh from 2007 to 2011, together with 264 publicly available HA sequences of clade 2.2, 2.3.2 and 2.3.4 retrieved from GenBank. Our study provides evidence that clade 2.2.2 viruses that caused outbreaks in Bangladesh were lineages independent from the viruses introduced earlier into India. Furthermore, the Bangladesh clade 2.2.2 descendents subsequently spread to India and Bhutan. This has implications for avian influenza control in southern Asia suggesting multiple routes of entry of the virus including one pathway that spread to neighboring countries via Bangladesh.