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Detection and isolation of highly pathogenic H5N1 avian influenza A viruses from blow flies collected in the vicinity of an infected poultry farm in Kyoto, Japan, 2004
Abstract and Figures
During the outbreak of highly pathogenic avian influenza that occurred in Tamba Town, Kyoto Prefecture in 2004, a total of 926 flies were collected from six sites within a radius of 2.3 km from the poultry farm. The H5 influenza A virus genes were detected from the intestinal organs, crop, and gut of the two blow fly species, Calliphora nigribarbis and Aldrichina grahami, by reverse transcription-polymerase chain reaction for the matrix protein (M) and hemagglutinin (HA) genes. The HA gene encoding multiple basic amino acids at the HA cleavage site indicated that this virus is a highly pathogenic strain. Based on the full-length sequences of the M, HA, and neuraminidase (NA) segments of virus isolates through embryonated chicken eggs, the virus from C. nigribarbis (A/blow fly/Kyoto/93/2004) was characterized as H5N1 subtype influenza A virus and shown to have > 99.9% identities in all three RNA segments to a strain from chickens (A/chicken/Kyoto/3/2004) and crows (A/crows/Kyoto/53/2004) derived during this outbreak period in Kyoto in 2004. Our results suggest it is possible that blow flies could become a mechanical transmitter of H5N1 influenza virus.
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Supplementary resources (3)
Nucleotide Sequence
April 2005
Nucleotide Sequence
April 2005
... Insects such as mosquitoes, blow flies and house flies are suggested as possible vectors of HPAIv. During a HPAI H5N1 epidemic on poultry farms in Japan, flies were collected from six sites within a radius of approximately 2 km from infected poultry farms [31]. HPAI H5N1 virus-identical to the virus strain derived from chickens, large-billed crows (Corvus macrorhynchos) and Black crows in the epidemic area-was detected in the intestinal organs, crop and gut of two blow fly species by RT-PCR, suggesting that blow flies can be a mechanical transmitter of HPAIv. ...
Wind-supported transport of particle matter (PM) contaminated with excreta from highly pathogenic avian influenza virus (HPAIv)-infected wild birds may be a HPAIv-introduction pathway, which may explain infections in indoor-housed poultry. The primary objective of our study was therefore to measure the nature and quantity of PM entering poultry houses via air-inlets. The air-inlets of two recently HPAIv-infected poultry farms (a broiler farm and a layer farm) were equipped with mosquito-net collection bags. PM was harvested every 5 days for 25 days. Video-camera monitoring registered wild bird visits. PM was tested for avian influenza viruses (AIV), Campylobacter and Salmonella with PCR. Insects, predominantly mosquitoes, were tested for AIV, West Nile, Usutu and Schmallenberg virus. A considerable number of mosquitoes and small PM amounts entered the air-inlets, mostly cobweb and plant material, but no wild bird feathers. Substantial variation in PM entering between air-inlets existed. In stormy periods, significantly larger PM amounts may enter wind-directed air-inlets. PM samples were AIV and Salmonella negative and insect samples were negative for all viruses and bacteria, but several broiler and layer farm PM samples tested Campylobacter positive. Regular wild (water) bird visits were observed near to the poultry houses. Air-borne PM and insects—potentially contaminated with HPAIv or other pathogens—can enter poultry air-inlets. Implementation of measures limiting this potential introduction route are recommended.
... Indeed, tsetse flies have been observed to feed on a broader range of mammals (including small mammals, Gaithuma et al., 2020) but also on birds and reptiles (e.g., Bitome-Essono et al., 2017;Sawabe et al., 2006). It is notable that one bird species was detected in the present study even though our primers are designed to be mammal specific. ...
Many emerging infectious diseases originate from wild animals, so there is a profound need for surveillance and monitoring of their pathogens. However, the practical difficulty of sample acquisition from wild animals tends to limit the feasibility and effectiveness of such surveys. Xenosurveillance, using blood‐feeding invertebrates to obtain tissue samples from wild animals and then detect their pathogens, is a promising method to do so. Here we describe the use of tsetse fly blood meals to determine (directly through molecular diagnostic and indirectly through serology), the diversity of circulating blood‐borne pathogens (including bacteria, viruses and protozoa) in a natural mammalian community of Tanzania. Molecular analyses of captured tsetse flies (182 pools of flies totalizing 1728 flies) revealed that the blood meals obtained came from 18 different vertebrate species including 16 non‐human mammals, representing approximately 25% of the large mammal species present in the study area. Molecular diagnostic demonstrated the presence of different protozoa parasites and bacteria of medical and/or veterinary interest. None of the six virus species searched for by molecular methods were detected but an ELISA test detected antibodies against African swine fever virus among warthogs, indicating that the virus had been circulating in the area. Sampling of blood‐feeding insects represents an efficient and practical approach to tracking a diversity of pathogens from multiple mammalian species, directly through molecular diagnostic or indirectly through serology, which could readily expand and enhance our understanding of the ecology and evolution of infectious agents and their interactions with their hosts in wild animal communities.
... In early spring and late autumn with low ambient temperature, A. grahami is usually the first species to colonize a corpse, and in certain extreme cases, the only colonizer (Guo et al. 2011). In addition, A. grahami can cause myiasis and can transmit pathogens such as H5N1 (Sawabe et al. 2006, Cao et al. 2015. ...
Many flies of Diptera are common entomological evidence employed in forensic investigation. Exploring the existence of inter- and intra-species genomic differences of forensically relevant insects is of great importance. Aldrichina grahami is a common blow fly species of forensic importance. The present study characterized the gene repertoires of A. grahami, and provides insights into issues related to forensic entomology, such as necrophagous behavior, gene family features, and developmental patterns. Gene families were clustered and classified according to their function in different aspects of the necrophagous lifestyle, generating several gene repertoires. The genes under positive selection pressure and evolutionary changes were screen and identified. Moreover, genes that exhibited potential prediction value in the post mortem interval (PMI) estimation and development of immature stages were subjected to analysis based on the developmental transcriptome. Related insect species were compared at the genomic level to reveal the genes associated with necrophagous behaviors. The expression of selected genes in separated repositories was verified using qPCR. This work was conducted using a high-quality chromosome-level genome assembly of A. grahami and its developmental transcriptome. The findings will facilitate future research on A. grahami and the other forensically important species.
... For example, one study from South Korea in 2006-07 found that outbreaks of HPAI in domestic poultry resembled the virus found in faecal samples from nearby migratory birds (Lee et al., 2008). The exact route of transmission from migratory birds to farmed birds is unclear, and could involve other birds, rodents or even insects (Sawabe et al., 2006). The emphasis on the role of migratory birds often overshadows the role of human transmission along the supply chain. ...
Outbreaks of avian influenza spread across political boundaries, and migratory birds are often scapegoated as vectors for the disease. This paper develops the vice of biosecurity to explain the tendency for conventional biosecurity to urge for greater restrictions on life that prove difficult to reverse and at times hazard increasing the risks arising from emerging infectious diseases. Drawing on a case study of avian influenza in Japan, this paper analyses the emergence of avian influenza and biosecurity responses to this disease. Avian influenza enacts a unique disease ecology that officials attempt to control by enforcing borders between Japanese/foreign territory, farmed/wild birds and healthy/infected life. Since facing heavy criticism for its initial responses, the Japanese state developed protocols for the swift testing and culling of chickens at infected farms. The worst season for avian influenza outbreaks occurred in the winter of 2020–21, which coincided with the Covid-19 pandemic. Through the vice of biosecurity, this paper draws attention to the problematic dynamic through which biosecurity measures are continually tightened as a first step in shifting towards a set of a biosecurity practices that are more accepting of the dynamism, diversity and mobility of life.
... Because C. grahami colonizes decomposing materials and is synanthrophropic, it can transmit human and animal pathogens from decomposing animals to individuals living in the surrounding area. One pathogen transmitted by this specie is the avian influenza virus (H5N1) (Tsuda et al. 2009, Sawabe et al. 2006. Other bacteria such as ...
Se presentan resultados de la sucesión ecológica de insectos asociados a cadaveres de Cerdos (Sus scrofa) en Saltillo, Coahuila así como el desarrollo de Phormia regina bajo condiciones de laboratorio y su validación en campo.
Conventional methods to study insect taxonomy especially forensic and medical dipterous flies are often tedious, time-consuming, labor-intensive, and expensive. An automated recognition system with image processing and computer vision provides an excellent solution to assist the process of insect identification. However, to the best of our knowledge, an image dataset that describes these dipterous flies is not available. Therefore, this paper introduces a new image dataset that is suitable for training and evaluation of a recognition system involved in identifying the forensic and medical importance of dipterous flies. The dataset consists of a total of 2876 images, in the input dimension (224 × 224 pixels) or as an embedded image model (96 × 96 pixels) for microcontrollers. There are three families (Calliphoridae, Sarcophagidae, Rhiniidae) and five genera (Chrysomya, Lucilia, Sarcophaga, Rhiniinae, Stomorhina), and each class of genus contained five different variants (same species) of fly to cover the variation of a species. Measurement(s)supervised machine learningTechnology Type(s)Camera DeviceSample Characteristic - OrganismDiptera sp. NZAC 03009335 Measurement(s) supervised machine learning Technology Type(s) Camera Device Sample Characteristic - Organism Diptera sp. NZAC 03009335
Brevibacillus laterosporus has entomopathogenic potential against several orders of insects and its wide bioactivity is associated with a variety of strain‐specific molecules. In order to avoid the use of synthetic insecticides, along with the need to control insect pests, microbial control has been widely used. Muscoid dipterans are known for their medical‐veterinary and sanitary importance, and synanthropy. The enormous biotechnological potential of B. laterosporus has been demonstrated, but there are still few studies with muscoid dipterans. The aim of the study was to verify the mortality of B. laterosporus NRS590 on synanthropic flies and to characterize its different cell stages ultrastructurally. The flies were collected from garbage bins and the colonies were adapted to the laboratory conditions. Bioassays with neo larvae were carried out from the bacterial growth in the phases: vegetative (6 hr), sporangium (20 hr), and free spores (44 hr). An aliquot of each phase was collected for Transmission (TEM) and Scanning Electron Microscopy (SEM). The effectiveness of NRS590 was observed in the sporulation phase, where the corrected mortality was 83.3, 85.1, and 99% for Chrysomya megacephala, Chrysomya putoria, and Musca domestica, respectively. The parasporal body was observed in detail on the entire spore surface. Although our knowledge of this bacterium is growing, it remains to be determined the real virulence factors responsible for the wide entomopathogenic activity observed on muscoid dipterans. Therefore, this study can provide subsidies for the improvement of efficient and safe microbial control techniques for the environment and living beings. Spores of NRS 590 against sinantropic muscoid dipterans; Ultrastructure of the different cell stages of NRS 590 using transmission and scanning electron microscopy techniques; NRS 590 has biotechnological potential as biopesticide.
This study elucidates the sufficient conditions for time-delayed scalar discrete models with nonlinear decimation terms to have at least N positive periodic solutions. Our results are proved using Krasnosel’skii’s fixed point theorem. Two sets, referred to as Δ and ∇, that are specifically determined for each given model play an important role in the results. The results obtained clarify that at least one positive periodic solution exists within the interval between a point belonging to Δ and a point belonging to ∇. Therefore, the number of disjoint intervals separated by multiple pairs determines the number of periodic solutions in the model. Although it is necessary to examine the individual discrete models in detail to identify whether the set Δ is present, the existence of the set ∇ is relatively easy to determine. Simple conditions for guaranteeing the existence of the set ∇ are also presented. In addition, a method of visual interpretation of our results for a special case of describing the population dynamics of adult flies is proposed. Finally, based on the results obtained, measures to effectively decimate adult flies are discussed, and it is shown that the decimation of adult flies will reduce the lower bound of the cycle change of the fly population but will not always reduce the upper bound.
The present opinion has the format of a risk profile and presents potential biological and chemical hazards as well as allergenicity and environmental hazards associated with farmed insects used as food and feed taking into account of the entire chain, from farming to the final product. The opinion also addresses the occurrence of these hazards in non-processed insects, grown on different substrate categories, in comparison to the occurrence of these hazards in other non-processed sources of protein of animal origin. When currently allowed feed materials are used as substrate to feed insects, the possible occurrence of microbiological hazards is expected to be comparable to their occurrence in other non-processed sources of protein of animal origin. The possible occurrence of prions in non-processed insects will depend on whether the substrate includes protein of human or ruminant origin. Data on transfer of chemical contaminants from different substrates to the insects are very limited. Substrates like kitchen waste, human and animal manure are also considered and hazards from insects fed on these substrates need to be specifically assessed. It is concluded that for both biological and chemical hazards, the specific production methods, the substrate used, the stage of harvest, the insect species and developmental stage, as well as the methods for further processing will all have an impact on the occurrence and levels of biological and chemical contaminants in food and feed products derived from insects. Hazards related to the environment are expected to be comparable to other animal production systems. The opinion also identifies the uncertainties (lack of knowledge) related to possible hazards when insects are used as food and feed and notes that there are no systematically collected data on animal and human consumption of insects. Studies on the occurrence of microbial pathogens of vertebrates as well as published data on hazardous chemicals in reared insects are scarce. Further data generation on these issues are highly recommended.
The minimum postmortem interval (PMImin) could be evaluated from the developmental stage of forensically important insects colonize a corpse, such as blow flies (Diptera: Calliphoridae). Unlike larvae, the developmental stage of which is well established according to their morphology, estimating the age of pupae is proven to be challenging. Recently, several studies reported the regulation of special genes during the development of blow fly pupae. However, gene regulation in Aldrichina grahami during the intrapuparial period remains to be studied. Therefore, we set out to investigate the mRNA levels of heat shock protein 23 (Hsp23), heat shock protein 24 (Hsp24), and 1_16 during the metamorphosis of A. grahami pupae. First, we examined seven candidate reference genes (ribosomal protein 49 (RP49), 18S ribosomal RNA (18S rRNA), 28S ribosomal RNA (28S rRNA), beta-tubulin at 56D (β-tubulin), Ribosomal protein L23 (RPL23), glutathione S-transferase (GST1), and Actin. Three widely used algorithms (NormFinder, BestKeeper, and geNorm) were applied to evaluate the mRNA levels of reference gene candidates in puparium at three stable temperatures (15, 22, and 27°C). Next, mRNA expression of Hsp23, Hsp24, and 1_16 during A. grahami metamorphosis was examined. We demonstrated that mRNA expression levels of Hsp23, Hsp24, and 1_16 showed time-specific regulation. In summary, our study identified three gene markers for the intrapuparial period of A. grahami and might provide a potential application in PMImin estimation.
Field study in Hachijo Island, Tokyo, showed Calliphora nigribarbis Vollenhoven was short-day univoltine insect whose development continued for 6 months (December to May). Adults appeared from November to June in towns (alt. 50m) and from October to July in the montane moss forests of Mt. Hachijo-Fuji (alt. 850m). The number of flies at the upland gradually increased during February to April and then decreased. After the rainy season in June, no adults were trapped throughout summer in both lowlands and uplands. No developing stages were also observed from June to November in both habitats. The first appearance of aged males and females in moss forests in October, suggests that adults aestivate there. After aestivation some females migrate from uplands to lowlands for spawning eggs. The lowland population emerged in spring migrates to uplands for aestivation. This up- and down seasonal migration was confirmed by the release and recapture of marked flies in the field experiment made during the same period. Adults seem to escape from lowlands in order to maintain diapause.
An investigation was made to know the seasonal fluctuation of Aldrichina grahami (Aldrich) in Aogashima Island, Tokyo, from December 1982 to December 1984. A trap baited with horse flesh was set at a station on a hill at 300m above sea level, and the numbers of flies trapped were counted once a month. Adult flies were captured from November to June or July, but none was seen for succeeding 3-4 months. It is not clear that females captured in autumn could aestivate in the island or migrated from the nearest Hachijo Island.
The breeding and emerging periods of Aldrichina grahami (Aldrich) were studied along with the fluctuation of adults for tracing its life cycle by using both baited and emergence traps in the backyard of the National Institute of Health, Tokyo, from December 1981 to March 1983. Seasonal activity of the adult in each year was similar, which confirmed the previous works reported from the same locality. A number of both sexes were trapped from March to July, and a large amount of females were caught in September to December. The first flight in early fall represents only aged females attracted. The breeding stage first appeared in the beginning of September and ended in the middle of coming May. Early fall generation derived from eggs deposited in September quickly grew up and emerged as adults in late fall (October to November). Prepupae or pupae of late fall generation derived from eggs in November to December became dormant during winter. After the dormancy, most adults emerged in March, though a few appeared in the end of January and the beginning of February. Spring generation from eggs in February to May normally developed within relatively short periods (0.5-1.5 months). No developing stages were observed from late May to early September, although male and female adults were trapped until the end of July. From the above, it is concluded that this blow fly can develop in the Tokyo area during 8.5 months from September to the middle of May excepting the coldest season in which dormant prepupae and pupae are dominant. The first appearance of aged females in September may suggest the adult aestivation in Aldrichina grahami.
An investigation was made to know the life history of Aldrichina grahami (Aldrich, 1930) in Hachijo Island, Tokyo, from February 1981 to May 1982. A trap baited with horse meat was set at five different altitudes, 50m, 200m, 400m, 600m and 800m, of Mt. Hachijo-Fuji. The number of flies captured was counted once a month. The larvae and pupae which had bred from meat in the trap were also counted and transferred into a separate trap for emergence. The number of the flies gradually increased from foot to top of the mountain during March to May. Following the rainy season in June, the number decreased and only one female was trapped at alt. 600m during August to September. Some females were captured again at alt. 600m and 800m in October. This suggests that some of mated females can estivate in the mountainous area above alt. 600m where it is usually cool and foggy during summer and mossforests develop. A few adults and a number of pupae were found even in winter in the trap of station 600m. The upper region of the mountain may be considered as a primary habitat of this blow fly. After estivation some females probably migrated for spawning eggs from the mountainous habitat and appeared in the town at alt. 50m. They may be successful in finding enough foods and breeding places around the human dwellings in winter and spring. The 74% of the flies were trapped in a temperature range of 2.0-9.0℃ in minimum and 16.5-23.0℃ in maximum and in a range of day length 10 : 30-14 : 20. This also suggests that the adult has a preference for such climate as being rather cool before sunrise and warm in daytime in spring and autumn.
Mark and recapture experiments of blow flies, Calliphora nigribarbis Vollenhoven and Aldrichina grahami (Aldrich), were attempted on Hachijo Island, Tokyo, in the autumn of 1983 and the spring of 1984 to investigate their seasonal migration. One female C. nigribarbis released at Station (St.) M (altitude 700m) in the moss forest of Mt. Hachijo-Fuji (854m) at the end of October 1983 was recaptured at St. 1 (50m) in the town at the end of November. At the end of March 1984,a total of 841 flies of both sexes of C. nigribarbis and A. grahami were marked and released at St. T (50m). One male and one female C. nigribarbis and one male A. grahami were recaptured at St. G (600m), St. 3 (400m) and St. 2 (200m), respectively, at the end of April. One female C. nigribarbis released on 24-25 May 1984 at St. T was trapped at St. 4 (600m) on 29 May 1984. The result of mark and recapture experiments confirmed the possibility that adults emerged in the town migrate from the lowland to the moss forest of Mt. Hachijo-Fuji before summer. Some of them most probably return to the lowland in autumn.
Eleven species of the calyptrate muscoid flies were captured on the ships which were located at the ocean weather stations or voyaged on the Pacific Ocean and East China Sea. Calliphora nigribarbis Vollenhoven and Aldrichina grahami (Aldrich) visited the weather ships at the stations which are located on the Pacific Ocean and East China Sea, 300 to 450km off Kyushu, Japan. Bengalia torosa (Wiedemann) was captured on a ship voyaged more than 100km from Taiwan where this fly is commonly found. These three species and Delia platura (Meigen) are considered to be transoceanic migratory insects, because they had more or less heavily damaged wings caused by a long-distant flight. The following 7 species, Lucilia cuprina, L. sericata, Musca domestica, Muscina stabulans, Atherigona varia, Lispe assimilis and Lispocephala miki, are considered to have moved with the ships, because they have had complete wings.
In the autumn of 1995,we had an opportunity to witness and evaluate the importance of the mass, probably transoceanic, migratory flight and landing of Calliphora nigribarbis Vollenhoven in the campus of Institute of Tropical Medicine, Nagasaki University, Nagasaki, Kyushu, western Japan. On October 25 (14 : 30 to 17 : 30,fine) and 26 (10 : 30 to 17 : 40,fine), they were seen flying in large numbers from the northwest to southeast across the top of the laboratory building (about 10m in height). They flew continuously in the direction of the wind and we caught some of them at the top of the building. They flew vigorously, with many flying well beyond the reach of our insect nets and without changing course. Some flies reaching the building in the evening landed on the concrete wall of the building. The migratory flights were simultaneously observed in Fukuoka where the flies were seen flying from the north across Tsushima-Kaikyo Strait. Fukuoka and Nagasaki are situated about 250 to 300km southeast of the Korean Penninsula. It could be inferred that these flies migrate across from the Asian continent to the main island Kyushu. C. nigribarbis has been considered to migrate altitudinally in the mainland Japan. However, the number of flies found in autumn (November) in Kyushu appears to be also increased by the transoceanic migration.
The calyptrate Diptera are relatively homogenous in their appearance compared to the Nematocera, being mostly robust flies with conspicuous bristles — in fact the epitome of the popular idea of flies′. The 18 000 or so described species form a natural group (′subsection′) within the higher Diptera (i.e. the infraorder Muscomorpha). Their classification is described in brief in Chapter 3.