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.

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 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.

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.

Comparative evaluation of four competitive/blocking ELISAs for the detection of influenza A antibodies in horses.

New Zealand is free from equine influenza and has never experienced an incursion in its horse population. As part of New Zealand's preparedness to an incursion of an exotic animal disease, it was considered necessary to select the most accurate test for equine influenza (EI) from the array of those available. Four readily available blocking/competitive enzyme-linked immunosorbent assays (ELISA), originally developed and marketed for the detection of antibodies against the avian influenza virus, were evaluated using serum samples from New Zealand non-infected, non-vaccinated horses (n=365), and Australian field infected (n=99) and experimentally infected horses (n=3). Diagnostic specificities (DSP) and diagnostic sensitivities (DSE) were determined as follows: ELISA-1=98.1%/99.0%; ELISA-2=90.1%/99.0%; ELISA-3=98.1%/96.0%; ELISA-4=95.3%/99.0%. For ELISA-1, DSP and DSE results were comparable to previously published data on a larger sample number from Australian horses (Sergeant et al., 2009). Receiver operating characteristics (ROC) and frequency histogram analysis were also performed. The area under the curve (AUC) ranged from 0.996 to 0.979, with ELISA-1 possessing the highest AUC, followed by ELISA-2, ELISA-4 and ELISA-3. Separation of the negative and the positive serum panel was best for ELISA-4, followed by ELISA-2, ELISA-1 and ELISA-3. In three experimentally infected horses, sero-positivity was detected between 7 and 9 days post-infection, with ELISA-4 being most sensitive, followed by ELISA-1, ELISA-2 and ELISA-3. Overall, the four ELISAs performed well in this evaluation but some differences were observed.

Analysis of Newcastle disease virus quasispecies and factors affecting the emergence of virulent virus.

Genome sequence analysis of a number of avirulent field isolates of Newcastle disease virus revealed the presence of viruses (within their quasispecies) that contained virulent F0 sequences. Detection of these virulent sequences below the ~1% level, using standard cloning and sequence analysis, proved difficult, and thus a more sensitive reverse-transcription real-time PCR procedure was developed to detect both virulent and avirulent NDV F0 sequences. Reverse-transcription real-time PCR analysis of the quasispecies of a number of Newcastle disease virus field isolates, revealed variable ratios (approximately 1:4-1:4,000) of virulent to avirulent viral F0 sequences. Since the ratios of these sequences generally remained constant in the quasispecies population during replication, factors that could affect the balance of virulent to avirulent sequences during viral infection of birds were investigated. It was shown both in vitro and in vivo that virulent virus present in the quasispecies did not emerge from the "avirulent background" unless a direct selection pressure was placed on the quasispecies, either by growth conditions or by transient immunosuppression. The effect of a prior infection of the host by infectious bronchitis virus or infectious bursal disease virus on the subsequent emergence of virulent Newcastle disease virus was examined.

Heterosubtypic anti-avian H5N1 influenza antibodies in intravenous immunoglobulins from globally separate populations protect against H5N1 infection in cell culture.

With antigenically novel epidemic and pandemic influenza strains persistently on the horizon it is of fundamental importance that we understand whether heterosubtypic antibodies gained from exposures to circulating human influenzas exist and can protect against emerging novel strains. Our studies of IVIG obtained from an infection-naive population (Australian) enabled us to reveal heterosubtypic influenza antibodies that cross react with H5N1. We now expand those findings for an Australian donor population to include IVIG formulations from a variety of northern hemisphere populations. Examination of IVIGs from European and South East-Asian (Malaysian) blood donor populations further reveal heterosubtypic antibodies to H5N1 in humans from different global regions. Importantly these protect against highly pathogenic avian H5N1 infection in vitro, albeit at low titres of inhibition. Although there were qualitative and quantitative differences in binding and protection between globally different formulations, the heterosubtypic antibody activities for the respective IVIGs were in general quite similar. Of particular note because of the relative geographic proximity to the epicentre of H5N1 and the majority of human infections, was the similarity in the antibody binding responses between IVIGs from the Malayan peninsula, Europe and Australia. These findings highlight the value of employing IVIGs for the study of herd immunity, and particularly heterosubtypic antibody responses to viral antigens such as those conserved between circulating human influenzas and emerging influenza strains such as H5N1. They also open a window into a somewhat ill defined arena of antibody immunity, namely heterosubtypic immunity.

A commercial ELISA detects high levels of human H5 antibody but cross-reacts with influenza A antibodies.

BACKGROUND AND OBJECTIVES: Commercial serological assays to determine influenza A H5N1 infection are available, although the accuracy and reproducibility of these are not reported in detail. This study aimed to assess the validity of a commercial ELISA H5 hemagglutinin (HA) antibody kit. STUDY DESIGN: A commercial ELISA for detection of antibodies towards influenza A H5 HA was evaluated using human sera from vaccinated individuals. The ELISA was used to screen 304 sera with elevated influenza A complement fixation titres collected between the period 1995-2007. RESULTS AND CONCLUSIONS: The ELISA was found to be accurate for sera with high levels of anti-H5 antibodies, and would be useful in clinical settings where a rapid result is required. Thirteen of the stored sera were positive using the ELISA, but were confirmed as negative for H5N1 exposure using further serological tests. Absorption studies suggested that antibodies towards seasonal H3N2 and H1N1 influenza may cross-react with H5 antigen, giving false positive results with the ELISA.

Surveillance for avian influenza in Nepal 2004-2005.

Highly pathogenic avian influenza has not been reported in Nepal to date. Surveillance for the presence of avian influenza viruses was conducted in 16 districts of Nepal from February 2004 to December 2005. Four hundred forty-six serum samples were collected from ducks, chickens, and pigeons and tested for antibodies to all influenza A viruses by competitive enzyme-linked immunosorbent assay (C-ELISA). Any sera positive by C-ELISA were tested for antibodies to H5, H7, and H9 influenza viruses by hemagglutination inhibition tests. One hundred and thirty-five cloacal swabs from healthy ducks and chickens were tested by commercial avian influenza antigen detection kits. A further 13 tissue samples from diseased birds were tested for the presence of virus by virus isolation in eggs, cell culture, and immunohistochemistry. No influenza viruses were detected in any of the tissues or swabs. All serum samples collected before October 2005 were negative for antibodies. The first sera positive for antibodies were collected on October 13, 2005, which were determined to be of the H9N2 subtype. This is the first report of serologic evidence of an avian influenza virus infection in Nepal.

Reduced sensitivity of influenza A (H5N1) to oseltamivir.

We tested the neuraminidase drug sensitivity of clade 1 and clade 2 influenza A (H5N1). All viruses demonstrated similar sensitivity to zanamivir, but compared with the 2004 clade 1 viruses, the Cambodian 2005 viruses were 6-fold less sensitive and the Indonesian clade 2 viruses were up to 30-fold less sensitive to oseltamivir.