Non-Human Primates Harbor Diverse Mammalian and Avian Astroviruses Including Those Associated with Human Infections.

Astroviruses (AstVs) are positive sense, single-stranded RNA viruses transmitted to a wide range of hosts via the fecal-oral route. The number of AstV-infected animal hosts has rapidly expanded in recent years with many more likely to be discovered because of the advances in viral surveillance and next generation sequencing. Yet no study to date has identified human AstV genotypes in animals, although diverse AstV genotypes similar to animal-origin viruses have been found in children with diarrhea and in one instance of encephalitis. Here we provide important new evidence that non-human primates (NHP) can harbor a wide variety of mammalian and avian AstV genotypes, including those only associated with human infection. Serological analyses confirmed that >25% of the NHP tested had antibodies to human AstVs. Further, we identified a recombinant AstV with parental relationships to known human AstVs. Phylogenetic analysis suggests AstVs in NHP are on average evolutionarily much closer to AstVs from other animals than are AstVs from bats, a frequently proposed reservoir. Our studies not only demonstrate that human astroviruses can be detected in NHP but also suggest that NHP are unique in their ability to support diverse AstV genotypes, further challenging the paradigm that astrovirus infection is species-specific.

Detection of a novel astrovirus from a black-naped monarch (Hypothymis azurea) in Cambodia.

BACKGROUND: Astroviruses are comprised of two genera with Avastrovirus infecting birds and Mamastrovirus infecting mammals. Avastroviruses have primarily been associated with infections of poultry, especially chicken, turkey, duck, and guineafowl production systems, but also infect wading birds and doves. Outcomes result in a spectrum of disease, ranging from asymptomatic shedding to gastroenteritis with diarrhea, stunting, failure to thrive and death. FINDINGS: Virological surveillance was conducted in birds from two sites in Cambodia in 2010. Samples were screened for influenza, astroviruses, coronaviruses, flaviviruses, and paramyxoviruses. A total of 199 birds were tested and an astrovirus was detected in a black-naped monarch (Hypothymis azurea). CONCLUSIONS: This is the first astrovirus detection in a passerine bird. Phylogenetic analysis and nucleotide distances suggest that this avastrovirus forms a distinct lineage and may constitute a fourth avastrovirus group.

Household practices related to disease transmission between animals and humans in rural Cambodia.

BACKGROUND: Zoonotic diseases are disproportionately affecting poor societies in low-income countries and pose a growing threat to public health and global food security. Rural Cambodian households may face an increased likelihood of exposure to zoonotic diseases as people there live in close association with livestock. The objectives of the study was to identify practices known to influence zoonosis transmission in rural Cambodian households and relate the practices to agro-ecological region, socio-economic position, demographics, livestock management and zoonosis awareness. METHODS: The study was conducted in three different agro-ecological regions of Cambodia; 10 villages each in the central lowlands, north-west wetlands and on the south coast, where information was obtained in questionnaires administered to 300 households, and 30 village heads and animal health workers. RESULTS: Descriptive analysis revealed a gender difference in responsibility for livestock and that the main purpose of raising livestock was for sale. Few respondents (6%) perceived a likelihood of disease transmission in their village between livestock, humans and wildlife, despite household practices related to zoonosis transmission being common. More than one-forth of households practised behaviours such as culling sick animals for consumption, eating animals found dead and allowing animals to enter sleeping and food preparation areas. Associations between household practices and possible explanatory factors were analysed with multivariable models using generalised estimation equations to account for clustering of practices within villages. Factors found to influence household practices were agro-ecological region, socio-economic position, number of people in the household, livestock species reared and awareness of zoonoses. CONCLUSIONS: Cambodia has experienced numerous fatal human cases of zoonotic influenza and extensive influenza information campaigns have been run, yet only a few of the households surveyed here reported the threat of zoonosis to be a concern in their village. Zoonosis awareness was positively related to hand washing behaviour, but other practices associated with an increased or decreased likelihood of exposure to zoonotic pathogens were unaffected by awareness. The findings indicate a knowledge-to-action gap among rural farmers and highlight the necessity for reconstructed interventions in zoonotic disease control.

Aerosol exposure of live bird market workers to viable influenza A/H5N1 and A/H9N2 viruses, Cambodia.

Live bird markets (LBMs) have been identified as key factors in the spread, persistence and evolution of avian influenza viruses (AIVs). In addition, these settings have been associated with human infections with AIVs of pandemic concern. Exposure to aerosolised AIVs by workers in a Cambodian LBM was assessed using aerosol impact samplers. LBM vendors were asked to wear an air sampler for 30 min per day for 1 week while continuing their usual activities in the LBM during a period of high AIV circulation (February) and a period of low circulation (May). During the period of high circulation, AIV RNA was detected from 100% of the air samplers using molecular methods and viable AIV (A/H5N1 and/or A/H9N2) was isolated from 50% of air samplers following inoculation into embryonated chicken eggs. In contrast, AIV was not detected by molecular methods or successfully isolated during the period of low circulation. This study demonstrates the increased risk of aerosol exposure of LBM workers to AIVs during periods of high circulation and highlights the need for interventions during these high-risk periods. Novel approaches, such as environmental sampling, should be further explored at key high-risk interfaces as a potentially cost-effective alternative for monitoring pandemic threats.

WildHealthNet: Supporting the development of sustainable wildlife health surveillance networks in Southeast Asia.

Wildlife and wildlife interfaces with people and livestock are essential surveillance targets to monitor emergent or endemic pathogens or new threats affecting wildlife, livestock, and human health. However, limitations of previous investments in scope and duration have resulted in a neglect of wildlife health surveillance (WHS) systems at national and global scales, particularly in lower and middle income countries (LMICs). Building on decades of wildlife health activities in LMICs, we demonstrate the implementation of a locally-driven multi-pronged One Health approach to establishing WHS in Cambodia, Lao PDR and Viet Nam under the WildHealthNet initiative. WildHealthNet utilizes existing local capacity in the animal, public health, and environmental sectors for event based or targeted surveillance and disease detection. To scale up surveillance systems to the national level, WildHealthNet relies on iterative field implementation and policy development, capacity bridging, improving data collection and management systems, and implementing context specific responses to wildlife health intelligence. National WHS systems piloted in Cambodia, Lao PDR, and Viet Nam engaged protected area rangers, wildlife rescue centers, community members, and livestock and human health sector staff and laboratories. Surveillance activities detected outbreaks of H5N1 highly pathogenic avian influenza in wild birds, African swine fever in wild boar (Sus scrofa), Lumpy skin disease in banteng (Bos javanicus), and other endemic zoonotic pathogens identified as surveillance priorities by local stakeholders. In Cambodia and Lao PDR, national plans for wildlife disease surveillance are being signed into legislation. Cross-sectoral and trans-disciplinary approaches are needed to implement effective WHS systems. Long-term commitment, and paralleled implementation and policy development are key to sustainable WHS networks. WildHealthNet offers a roadmap to aid in the development of locally-relevant and locally-led WHS systems that support the global objectives of the World Organization for Animal Health's Wildlife Health Framework and other international agendas.

Influenza virus infection in nonhuman primates.

To determine whether nonhuman primates are infected with influenza viruses in nature, we conducted serologic and swab studies among macaques from several parts of the world. Our detection of influenza virus and antibodies to influenza virus raises questions about the role of nonhuman primates in the ecology of influenza.

New outbreaks of H5N1 highly pathogenic avian influenza in domestic poultry and wild birds in Cambodia in 2011.

Five outbreaks of H5N1 highly pathogenic avian influenza (HPAI) have been diagnosed in domestic poultry and wild birds in Cambodia from January to November of 2011. Of the five outbreaks, one occurred in a village backyard flock in Kandal province in January; two occurred in native Cambodian chickens and ducks in Banteay Meanchey province in July and August, respectively; one was seen in wild birds in Phnom Tamao Zoo in Kandal Province in July; and one outbreak occurred in commercial broilers at Opong Moan in Battambang province in northwestern Cambodia in early November. Clinically, HPAI-infected broilers and native chickens showed sudden death, severe depression, ruffled feathers, edema of heads and necks, swollen and cyanotic combs and wattles, and swollen and congested conjunctiva, with occasional hemorrhage, paralysis, and other neurologic signs. In ducks, significantly swollen sinuses and eyes, cloudy corneas, difficulty standing, or paralysis were commonly seen. Some affected ducks showed sudden death without obvious clinical symptoms. Necropsy lesions showed congestion and necrotic debris within sinuses and severe hemorrhages in gizzards, livers, and lungs in both affected native chickens and ducks during the new outbreaks in 2011. All five outbreaks were diagnosed as H5N1 HPAI by virus isolation and real-time reverse transcription-PCR tests. Once a backyard flock in a village or a poultry farm was diagnosed as positive for H5N1 HPAI; the whole village backyard poultry and all farm flocks were culled immediately by Cambodian provincial and central authorities as per the strategies adopted for the control of HPAI.

Seroprevalence and awareness of porcine cysticercosis across different pig production systems in south-central Cambodia.

BACKGROUND: Taeniasis/cysticercosis, caused by the pork tapeworm Taenia solium, represents an important public health and economic burden in endemic countries. However, there is a paucity of data on infection among pigs in many parts of Southeast Asia, particularly Cambodia. We aimed to estimate seroprevalence of porcine cysticercosis, and investigate husbandary practices and knowledge of the disease among livestock workers, across different pig sector units in south-central Cambodia. METHODS: A cross sectional survey was conducted among pig smallholders, commercial farms, slaughterhouses and traders/middlemen from south-central Cambodia, selected through multistage sampling in proportion to local pig populations sizes. Questionnaires were administered to 163 pig workers to obtain data pig production, trading and slaughtering practices. Sera from 620 pigs were tested for Taenia antigens using a commercial ELISA-based test. Associations between seroprevalence and pig husbandry practices were assessed using generalised linear mixed models, adjusting for random-effects at herd-level. RESULTS: Of 620 pigs sampled, 29 (4.7%) tested positive for Taenia antigens. Seropositivity was associated with type of pig sector unit (P = 0.008), with the highest seroprevalence among pigs sampled from traders/middlemen (16.7%; 95% CI: 4.4%-37.8%), smallholders (7.6%; 95% CI: 3.8%-14.1%) and slaughterhouses (4.1%; 95% CI: 2.0%-7.5%), while none of the pigs sampled from small/medium or large commercial farms tested positive. Although the vast majority of pigs were penned, practices that might facilitate human-to-pig transmission, such as use of household waste and surface water sources to feed pigs, were prevalent among smallholders. However these were not found to be significantly associated with infection. Of 163 interviewed pig workers, 115 (70.5%) were aware of porcine cysticercosis, and 78 (47.8%) also knew it could affect humans. Twenty-six (16.0%) reported having noticed lesions typical of cysticercosis in their pigs. CONCLUSIONS: Despite most pigs being kept confined in pens rather than raised in free-roaming systems, porcine cysticercosis appears to be endemic in south-central Cambodia and is associated with smallholder production. Further investigation is needed to identify which Taenia species are causing infections among pigs, and how seroprevalence and zoonotic risk may vary across the country, to understand the risks to public health and assess where interventions might be needed.

Manure management and public health: Sanitary and socio-economic aspects among urban livestock-keepers in Cambodia.

Livestock manure is a valuable source of nutrients for crop production, but can also pose a public health hazard and have negative environmental impacts. This study investigated manure management practices among urban and peri-urban livestock keepers in Cambodia, to identify risk behaviours and socio-economic aspects associated with the handling of manure. A survey including 204 households was conducted, using a structured questionnaire with questions on demographics, socio-economic characteristics and household practices related to manure management. Faecal samples were obtained from pig pens and pig manure storage units for analysis of the potential zoonotic pathogens Salmonella enterica (Polymerase Chain Reaction (PCR)), Ascaris suum and Trichuris suis (McMaster flotation technique). The survey revealed a difference in management between cattle and pig manure. Cattle manure was most commonly used as fertiliser for crop production (66%) (p<0.001), whereas pig manure was most commonly dumped in the environment (46%) (p<0.001). Logistic regression models showed that households with a lower socio-economic position were more likely to dump pig manure (p<0.001), with scarcity of agricultural land (p<0.001) and lack of carts for transportation of manure (p<0.01) being identified as contributing factors. Salmonella enterica was detected in 9.7% of manure samples, while Ascaris suum and Trichuris suis were detected in 1.6% and 2.4% of the samples, respectively. The results presented in this study indicate that manure management by urban and peri-urban households may pose a public health threat and an environmental hazard. There is evidently a need for further knowledge support to the livestock keepers to promote good management practices.

Urban and peri-urban family-based pig-keeping in Cambodia: Characteristics, management and perceived benefits and constraints.

Keeping pigs in urban and peri-urban areas may not only provide many benefits for the urban households, but may also be challenging and a potential health hazard. The aim of this cross-sectional study was to describe household characteristics and to evaluate perceived benefits and constraints among pig-keepers in the urban and peri-urban areas of Phnom Penh, Cambodia. The study included 204 households and a structured questionnaire was used to interview the household member responsible for taking care of the pigs. Descriptive analyses showed that most households kept between 5 and 15 pigs and that all households kept their pigs in confinement. About 97% of the households owned the pigs themselves and the pigs were generally managed by female household members (43%). Pigs were mainly kept for commercial purposes and more than 60% of the households stated that income from pig-keeping was the main or one of the main sources of revenue for the household. More than 82% reported that they had experienced disease outbreaks among their pigs during the past three years and disease outbreaks were more commonly reported in households with lower socio-economic position (P = 0.025). Disease outbreaks were considered one of the main constraints, along with expensive feed and low payment prices for the slaughter pigs, but few households considered sanitary or other public health issues problematic. Thus, pig-keeping makes an important contribution to the livelihoods of urban and peri-urban households, but many households face external constraints on their production, such as diseases and low revenues, which may have a negative impact on their livelihoods.

Diversity of bat astroviruses in Lao PDR and Cambodia.

Astroviruses are known to infect humans and a wide range of animal species, and can cause gastroenteritis in their hosts. Recent studies have reported astroviruses in bats in Europe and in several locations in China. We sampled 1876 bats from 17 genera at 45 sites from 14 and 13 provinces in Cambodia and Lao PDR respectively, and tested them for astroviruses. Our study revealed a high diversity of astroviruses among various Yangochiroptera and Yinpterochiroptera bats. Evidence for varying degrees of host restriction for astroviruses in bats was found. Furthermore, additional Pteropodid hosts were detected. The astroviruses formed distinct phylogenetic clusters within the genus Mamastrovirus, most closely related to other known bat astroviruses. The astrovirus sequences were found to be highly saturated indicating that phylogenetic relationships should be interpreted carefully. An astrovirus clustering in a group with other viruses from diverse hosts, including from ungulates and porcupines, was found in a Rousettus bat. These findings suggest that diverse astroviruses can be found in many species of mammals, including bats.

Genetic diversity of coronaviruses in bats in Lao PDR and Cambodia.

South-East Asia is a hot spot for emerging zoonotic diseases, and bats have been recognized as hosts for a large number of zoonotic viruses such as Severe Acute Respiratory Syndrome (SARS), responsible for acute respiratory syndrome outbreaks. Thus, it is important to expand our knowledge of the presence of viruses in bats which could represent a risk to humans. Coronaviruses (CoVs) have been reported in bat species from Thailand, China, Indonesia, Taiwan and the Philippines. However no such work was conducted in Cambodia or Lao PDR. Between 2010 and 2013, 1965 bats were therefore sampled at interfaces with human populations in these two countries. They were tested for the presence of coronavirus by consensus reverse transcription-PCR assay. A total of 93 samples (4.7%) from 17 genera of bats tested positive. Sequence analysis revealed the presence of potentially 37 and 56 coronavirus belonging to alpha-coronavirus (αCoV) and beta-CoV (ßCoV), respectively. The ßCoVs group is known to include some coronaviruses highly pathogenic to human, such as SARS-CoV and MERS-CoV. All coronavirus sequences generated from frugivorous bats (family Pteropodidae) (n=55) clustered with other bat ßCoVs of lineage D, whereas one coronavirus from Pipistrellus coromandra fell in the lineage C of ßCoVs which also includes the MERS-CoV. αCoVs were all detected in various genera of insectivorous bats and clustered with diverse bat αCoV sequences previously published. A closely related strain of PEDV, responsible for severe diarrhea in pigs (PEDV-CoV), was detected in 2 Myotis bats. We highlighted the presence and the high diversity of coronaviruses circulating in bats from Cambodia and Lao PDR. Three new bat genera and species were newly identified as host of coronaviruses, namely Macroglossus sp., Megaerops niphanae and Myotis horsfieldii.

Detection of Campylobacter in human and animal field samples in Cambodia.

Campylobacter are zoonotic bacteria and a leading cause of human gastroenteritis worldwide with Campylobacter jejuni and C. coli being the most commonly detected species. The aim of this study was to detect Campylobacter in humans and livestock (chickens, ducks, pigs, cattle, water buffalo, quail, pigeons and geese) in rural households by routine culturing and multiplex PCR in faecal samples frozen before analysis. Of 681 human samples, 82 (12%) tested positive by PCR (C. jejuni in 66 samples and C. coli in 16), but none by routine culture. Children were more commonly Campylobacter positive (19%) than adult males (8%) and females (7%). Of 853 livestock samples, 106 (12%) tested positive by routine culture and 352 (41%) by PCR. Campylobacter jejuni was more frequent in chickens and ducks and C. coli in pigs. In conclusion, Campylobacter proved to be highly prevalent by PCR in children (19%), ducks (24%), chickens (56%) and pigs (72%). Routine culturing was insufficiently sensitive in detecting Campylobacter in field samples frozen before analysis. These findings suggest that PCR should be the preferred diagnostic method for detection of Campylobacter in humans and livestock where timely culture is not feasible.

Knowledge, attitudes and practices of rabies prevention and dog bite injuries in urban and peri-urban provinces in Cambodia, 2009.

Rabies remains a major public health issue despite the existence of well established prevention and treatment protocols. Knowledge and methods of practice were evaluated in an urban and peri-urban province of Cambodia (Phnom Penh and Kandal, respectively). The majority of respondents (93.2%; 233/250) had heard of the disease rabies, of whom only 77.3% (180/233) knew it was fatal to humans. In addition, only 51.9% (121/233) were aware of the vaccine for dogs. The proportion of the population that reported a dog bite (2004-2009) was similar for Phnom Penh and Kandal. Nearly one-half of all victims (37/75) sought treatment at the Institut Pasteur-Cambodia (IPC) clinic, followed by a private clinic (19/75), a hospital (6/75) and traditional medicine (4/75); 7 victims sought no treatment and 2 reported other. Overall, children aged <15 years reported a significantly greater proportion of dog bite victims than adults aged ≥15 years [10.0% (28/280) vs 4.4% (47/1059), respectively]. Nearly all dog owners agreed to pay for their dogs' vaccination (96.5%; 136/141) and to use a collar (94.3%; 133/141). Only 41.8% (59/141) and 51.8% (73/141) would pay to have their dogs spayed and neutered, respectively. Further community education on the prevention of rabies transmission is needed. Focusing on responsible dog ownership and the importance of both the prevention and immediate treatment following a dog bite is essential to reduce rabies infection in Cambodia.

Identifying live bird markets with the potential to act as reservoirs of avian influenza A (H5N1) virus: a survey in northern Viet Nam and Cambodia.

Wet markets are common in many parts of the world and may promote the emergence, spread and maintenance of livestock pathogens, including zoonoses. A survey was conducted in order to assess the potential of Vietnamese and Cambodian live bird markets (LBMs) to sustain circulation of highly pathogenic avian influenza virus subtype H5N1 (HPAIV H5N1). Thirty Vietnamese and 8 Cambodian LBMs were visited, and structured interviews were conducted with the market managers and 561 Vietnamese and 84 Cambodian traders. Multivariate and cluster analysis were used to construct a typology of traders based on their poultry management practices. As a result of those practices and large poultry surplus (unsold poultry reoffered for sale the following day), some poultry traders were shown to promote conditions favorable for perpetuating HPAIV H5N1 in LBMs. More than 80% of these traders operated in LBMs located in the most densely populated areas, Ha Noi and Phnom Penh. The profiles of sellers operating at a given LBM could be reliably predicted using basic information about the location and type of market. Consequently, LBMs with the largest combination of risk factors for becoming virus reservoirs could be easily identified, potentially allowing control strategies to be appropriately targeted. These findings are of particular relevance to resource-scarce settings with extensively developed LBM systems, commonly found in South-East Asia.

A(H5N1) Virus Evolution in South East Asia.

Highly Pathogenic Avian Influenza (HPAI) H5N1 virus is an ongoing public health and socio-economic challenge, particularly in South East Asia. H5N1 is now endemic in poultry in many countries, and represents a major pandemic threat. Here, we describe the evolution of H5N1 virus in South East Asia, the reassortment events leading to high genetic diversity in the region, and factors responsible for virus spread. The virus has evolved with genetic variations affecting virulence, drug-resistance, and adaptation to new host species. The constant surveillance of these changes is of primary importance in the global efforts of the scientific community.

Poultry movement networks in Cambodia: implications for surveillance and control of highly pathogenic avian influenza (HPAI/H5N1).

Movement of poultry through markets is potentially important in the circulation and spread of highly pathogenic avian influenza. However little is understood about poultry market chains in Cambodia. We conducted a cross-sectional survey of 715 rural villagers, 123 rural, peri-urban and urban market sellers and 139 middlemen from six provinces and Phnom Penh, to evaluate live poultry movement and trading practices. Direct trade links with Thailand and Vietnam were identified via middlemen and market sellers. Most poultry movement occurs via middlemen into Phnom Penh making live bird wet markets in Phnom Penh a potential hub for the spread of H5N1 and ideal for surveillance and control.

Changes in poultry handling behavior and poultry mortality reporting among rural Cambodians in areas affected by HPAI/H5N1.

BACKGROUND: Since 2004, 21 highly pathogenic avian influenza H5N1 outbreaks in domestic poultry and eight human cases have been confirmed in Cambodia. As a result, a large number of avian influenza education campaigns have been ongoing in provinces in which H5N1outbreaks have occurred in humans and/or domestic poultry. METHODOLOGY/PRINCIPAL FINDINGS: Data were collected from 1,252 adults >15 years old living in two southern provinces in Cambodia where H5N1 has been confirmed in domestic poultry and human populations using two cross-sectional surveys conducted in January 2006 and in November/December 2007. Poultry handling behaviors, poultry mortality occurrence and self-reported notification of suspect H5N1 poultry cases to animal health officials in these two surveys were evaluated. Our results demonstrate that although some at risk practices have declined since the first study, risky contact with poultry is still frequent. Improved rates of reporting poultry mortality were observed overall, but reporting to trained village animal health workers decreased by approximately 50%. CONCLUSIONS/SIGNIFICANCE: Although some improvements in human behavior have occurred, there are still areas--particularly with respect to the handling of poultry among children and the proper treatment of poultry and the surrounding household environment--that need to be addressed in public health campaigns. Though there were some differences in the sampling methods of the 2006 and 2007 surveys, our results illustrate the potential to induce considerable, potentially very relevant, behavioral changes over a short period of time.