Avian Samples Research Articles

Winged odyssey: Profiling bacterial vistas in migratory avifauna via 16S rRNA sequencing

Avian migration is an intrinsic biological phenomenon that involves trans-boundary movements to evade adverse ecological circumstances. During migration, avian gut bacterial taxa may serve as a potential source of bacterial dissemination via fecal contamination at stop-over sites. Therefore, bacterial taxa composition as well as diversities were investigated employing 16S rRNA sequencing in fecal samples collected from flocks of seven migratory avian species visiting southern districts of Khyber Pakhtunkhwa, Pakistan. The analysis revealed that Grus virgo exhibits the highest alpha diversity, followed by Aythya ferina while G. grus reflects lowest diversity among all the migratory avian fecal samples. The findings depicted significant variations in the bacterial beta diversities of migratory avifauna. At phylum level, Firmicutes, Proteobacteria, and Actinobacteriota showed the highest relative abundance in Plegadis falcinellus, Chlamydotis undulata and Aythya ferina respectively. Further exploration within phyla elucidates finer-scale taxonomic differences at the family and genus levels. This study identified potential pathogenic bacteria such as Staphylococcus, Streptococcus, Enterococcus, Proteus, Clostridium sensu stricto 1, Fusobacterium and Escherichia that offers valuable insight into the microbiological hazards associated with migratory birds. Although pathogenicity was not directly assessed, the observed relative abundance of opportunistic bacterial genera suggests continuous surveillance of gut bacterial community during migration to safeguard avian biodiversity and mitigate escalating threats of infection emergence and dissemination.

Retrospective study on the occurrence of Salmonella serotypes in veterinary specimens of Atlantic Canada (2012-2021).

This study aimed to summarize the frequency and the antimicrobial susceptibility profiles of the Salmonella serotypes identified from the specimens of companion animals, livestock, avian, wildlife and exotic species within Atlantic Canada. The retrospective electronic laboratory data of microbiological analyses of a selected subset of samples from 03 January 2012 to 29 December 2021 submitted from various animal species were retrieved. The frequency of Salmonella serotypes identified, and their antimicrobial susceptibility results obtained using the disk diffusion or broth method were analysed. The test results were interpreted according to the Clinical and Laboratory Standards Institute standard. The Salmonella serotypes were identified by slide agglutination (Kauffman-White-Le-Minor Scheme) and/or the Whole Genome Sequencing for the Salmonella in silico Serovar Typing Resource-based identification. Of the cases included in this study, 4.6% (n=154) had at least one Salmonella isolate, corresponding to 55 different serovars. Salmonella isolation was highest from exotic animal species (n=40, 1.20%), followed by porcine (n=26, 0.78%), and canine (n=23, 0.69%). Salmonella subsp. enterica serovar Typhimurium was predominant among exotic mammals, porcine and caprine samples, whereas S. Enteritidis was mostly identified in bovine and canine samples. S. Typhimurium of porcine origin was frequently resistant (>70.0%) to ampicillin. In contrast, S. Typhimurium isolates from porcine and caprine samples were susceptible (>70.0%) to florfenicol. S. Oranienburg from equine samples was susceptible to chloramphenicol, but frequently resistant (>90.0%) to azithromycin. In avian samples, S. Copenhagen was susceptible (>90.0%) to florfenicol, whereas Muenchen was frequently resistant (>90.0%) to florfenicol. S. subsp. diarizonae serovar IIIb:61:k:1,5 of ovine origin was resistant (50.0% isolates) to sulfadimethoxine. No significant changes were observed in the antibiotic resistance profiles across the study years. This report provides data for surveillance studies, distribution of Salmonella serotypes and their antimicrobial resistance among veterinary specimens of Atlantic Canada.

Genetic Diversity of Avian Influenza Viruses Detected in Waterbirds in Northeast Italy Using Two Different Sampling Strategies.

Avian influenza viruses (AIVs), which circulate endemically in wild aquatic birds, pose a significant threat to poultry and raise concerns for their zoonotic potential. From August 2021 to April 2022, a multi-site cross-sectional study involving active AIV epidemiological monitoring was conducted in wetlands of the Emilia-Romagna region, northern Italy, adjacent to densely populated poultry areas. A total of 129 cloacal swab samples (CSs) and 407 avian faecal droppings samples (FDs) were collected, with 7 CSs (5.4%) and 4 FDs (1%) testing positive for the AIV matrix gene through rRT-PCR. A COI-barcoding protocol was applied to recognize the species of origin of AIV-positive FDs. Multiple low-pathogenic AIV subtypes were identified, and five of these were isolated, including an H5N3, an H1N1, and three H9N2 in wild ducks. Following whole-genome sequencing, phylogenetic analyses of the hereby obtained strains showed close genetic relationships with AIVs detected in countries along the Black Sea/Mediterranean migratory flyway. Notably, none of the analyzed gene segments were genetically related to HPAI H5N1 viruses of clade 2.3.4.4b isolated from Italian poultry during the concurrent 2021-2022 epidemic. Overall, the detected AIV genetic diversity emphasizes the necessity for ongoing monitoring in wild hosts using diverse sampling strategies and whole-genome sequencing.

Molecular evidence of Bartonella spp. in tropical wild birds from the Brazilian Pantanal, the largest wetland in South America.

Despite the worldwide occurrence of bartonellae in a broad range of mammal species, in which they usually cause a long-lasting erythrocytic bacteremia, few studies reported Bartonella spp. in avian hosts. The present work aimed to investigate the occurrence and molecular identity of Bartonella spp. infecting birds in the Pantanal wetland, central-western Brazil using a multigene approach. For this purpose, blood samples were collected from 517 individuals from 13 avian orders in the states of Mato Grosso and Mato Groso do Sul. DNA was extracted from avian blood and 500/517 (96.7%) samples were positive in a conventional PCR targeting the avian β-actin gene. Nineteen (3.8%) out of 500 avian blood samples were positive in a qPCR assay for Bartonella spp. based on the nuoG gene. Among 19 avian blood DNA samples positive in the qPCR for Bartonella spp., 12 were also positive in the qPCR for Bartonella based on the 16S-23S RNA Intergenic region (ITS). In the PCR assays performed for molecular characterization, one 16S rRNA, three ribC, and one nuoG sequences were obtained. Based on BLASTn results, while 1 nuoG, 2 ribC, and 2 ITS sequences showed high identity to Bartonella henselae, one 16S rRNA and 2 ITS showed high similarity to Bartonella machadoae in the sampled birds. Bartonella spp. related to B. henselae and B. machadoae were detected, for the first time, in wild birds from the Brazilian Pantanal.

Mitochondrial and apicoplast genome copy abundances of haemosporidian parasites are explained by host species and parasitic lineage

Endosymbiotic organelles, such as mitochondria and plastids, contain own remnant genomes (nucleoids), whose variable abundance in cells may be adaptive to the physiological necessities and functions of the cells. Unicellular apicomplexan parasites contain one mitochondrium and one apicoplast with variable genome copy numbers. We measured the abundance of mitochondrial, apicoplast and nuclear genome copies in a set of avian blood samples infected with haemosporidian blood parasites, belonging to the three main genera Plasmodium, Haemoproteus and Leucocytozoon. We designed general primers suitable for qPCR, amplifying fragments of the mitogenome, plastome and nuclear genomes of avian malaria and related haemosporidian parasites. We measured the amplification of these fragments in 153 samples of 23 avian host species and infected with 33 parasitic lineages. We estimate on average several hundred mitochondrial genome copies and several tens of apicoplast copies per haploid gametocyte cell with substantial variation among samples. Host species appeared to differ in their mitogenome abundance while parasitic lineages differed in plastome abundance per cell (per nuclear copy signal). We did not find consistent differences between parasite genera or higher avian taxa. Parasite lineages and host bird species did not differ consistently in infection intensity, estimated from parasite to host nuclear signals, which may indicate that samples were taken at different stages of infection. However, this and similar results remain to be cross-validated with in-situ imaging techniques. The novel molecular tools introduced here offer avenues for the characterization of nucleoid abundance of haemosporidian parasites over environmental conditions and parasitic developmental stages. Such measures will improve our understanding of parasite physiology, ecology, the coadaptation and coevolution with hosts and suggest possible augmentations to standard methods in the research field.

Prevalence of parasites in selected captive bird species.

Blood and fecal samples of chukar partridge (Alectoris chukar), albino pheasant (Phasianus colchicus), silver pheasant (Lophura nycthemera), rose-ringed parakeet (Psittacula krameri) and turkeys (Meleagris gallopavo) were analyzed to check parasitic prevalence. To record parasites these five avian species were placed kept in separate cages at Avian Conservation and Research Center, Department of Wildlife an Ecology, University of Veterinary and Animal Sciences, Lahore, Pakistan. 100 fecal and 100 blood samples for each bird species were inspected to analyze internal parasites. During present study, 17 species of endoparasites 14 from fecal samples and three from blood were examined. Two species of ectoparasites i.e. mite Dermanyssus gallinae 42% and fowl ticks Args persicus 41%were studied. Blood parasites included Plasmodium juxtanucleare 50%, Leucoctoyzoon simond having parasitic prevalence 40%, and Aegyptinella pullorum having parasitic prevalence of 40%. Parasitic species recorded from fecal samples included 6 species of nematodes viz. Allodpa suctoria 2%. Syngamus trachea with parasitic prevalence of 60%, Capillaria annulata 37.5%, Ascardia galli 24%, Capillaria anatis 40% and Heterakis gallinarum 28.3%. Similarly, two species of trematodes viz. Prosthogonimus ovatus having parasitic prevalence of 50% and Prosthogonimus macrorchis 21% were also documented from fecal avian samples . Single cestode species Raillietina echinobothrida having parasitic prevalence of 72% and 3 protozoan species i.e. Eimeria maxima having parasitic prevalence of 21%, Giardia lamblia 41% and Histomonas meleagridis 18% were documented during corpological analysis. In our recommendation, proper sanitation, medication and vaccination of bird's enclousres are suggested to avoid parasites.

Next-Generation Sequencing for the Detection of Microbial Agents in Avian Clinical Samples.

Direct-targeted next-generation sequencing (tNGS), with its undoubtedly superior diagnostic capacity over real-time PCR (RT-PCR), and direct-non-targeted NGS (ntNGS), with its higher capacity to identify and characterize multiple agents, are both likely to become diagnostic methods of choice in the future. tNGS is a rapid and sensitive method for precise characterization of suspected agents. ntNGS, also known as agnostic diagnosis, does not require a hypothesis and has been used to identify unsuspected infections in clinical samples. Implemented in the form of multiplexed total DNA metagenomics or as total RNA sequencing, the approach produces comprehensive and actionable reports that allow semi-quantitative identification of most of the agents present in respiratory, cloacal, and tissue samples. The diagnostic benefits of the use of direct tNGS and ntNGS are high specificity, compatibility with different types of clinical samples (fresh, frozen, FTA cards, and paraffin-embedded), production of nearly complete infection profiles (viruses, bacteria, fungus, and parasites), production of "semi-quantitative" information, direct agent genotyping, and infectious agent mutational information. The achievements of NGS in terms of diagnosing poultry problems are described here, along with future applications. Multiplexing, development of standard operating procedures, robotics, sequencing kits, automated bioinformatics, cloud computing, and artificial intelligence (AI) are disciplines converging toward the use of this technology for active surveillance in poultry farms. Other advances in human and veterinary NGS sequencing are likely to be adaptable to avian species in the future.

Evaluation of virulence factors in clinical isolates of pathogenic E. coli in avian samples in Caloto, Colombia

Evaluation of virulence factors in clinical isolates of pathogenic E. coli in avian samples in Caloto, Colombia

Development of a recombinase-aided amplification combined with a lateral flow dipstick assay for rapid detection of H7 subtype avian influenza virus.

Avian influenza viruses (AIV) pose a significant persistent threat to the public health and safety. It is estimated that there have been over 100 outbreaks caused by various H7 subtypes of avian influenza viruses (AIV-H7) worldwide, resulting in over 33 million deaths of poultry. In this study, we developed a recombinase-aided amplification combined with a lateral flow dipstick assay for the detection of hemagglutinin (HA) genes to provide technical support for rapid clinical detection of AIV-H7. The results showed that the assay can complete the reaction within 30 min at a temperature of 39°C. Specificity tests demonstrated that there was no cross-reactivity with other common poultry pathogens, including Newcastle disease virus (NDV) and infections bronchitis virus (IBV). The detection limit of this assay was 1 × 101 copies/μL, while RT-qPCR method was 1 × 101 copies/μL, and RT-PCR was 1 × 102 copies/μL. The κ value of the RT-RAA-LFD and RT-PCR assay in 132 avian clinical samples was 0.9169 (p < 0.001). These results indicated that the developed RT-RAA-LFD assay had good specificity, sensitivity, stability and repeatability and may be used for rapid detection of AIV-H7 in clinical diagnosis.

Surveillance on avian influenza and new castle disease in rift valley water body areas of Ethiopia

HPAI (High Pathogenic Avian Influenza) is one of the most serious public-health threats to arise from the animal reservoir. The diseases can kill up to 90% of a flock and generate epidemics that can spread quickly, wreaking havoc on the poultry business and putting severe trade restrictions in place. New Castle disease (NCD) is also a persistent threat to the poultry business and is a limiting illness for poultry farmers around the world. It is one of the most serious diseases in Ethiopia, with high mortality and huge economic losses. For this and the like purposes risk-based surveillance was conducted in selected sites in Oromia, Amhara, and Southern Nation Nationality Peoples Region (SNNPR) rift valley water body areas and 5-10 km radius districts from January to April 2019. The goal was to assess the presence or absence of Highly Pathogenic Avian Influenza (HPAI) and New Castle Disease (NCD) in migratory birds and domestic chicken in water body areas, as well as to assess the level of community awareness in the study areas. Total 6866 samples were collected, including 819 sera samples and 1836 tracheal and cloacal swab samples from backyard domestic chickens within a 5-10 km radius of rift valley lakes, and 4211 fecal droplet samples from wild/migratory birds. Avian influenza serum samples tested by HA/HI and Fecal droplet samples tested by RT PCR came back negative. The results for NDV swab 1336 samples tested by RT PCR were 182 samples positive, and 819 samples tested by HI were 72 samples positive. In the public health aspect, a questionnaire survey was completed by 180 members of the respective community. As a result, the respondents have a solid understanding of the migratory birds that visit their area.

Pathognomonic features of Pasteurella multocida isolates among various avian species in Sharkia Governorate, Egypt

The present study aimed to isolate Pasteurella multocida (P. multocida) from pulmonary cases in several avian species and then investigate the histopathological features, antimicrobial resistance determinants, virulence characteristics, and risk factors analysis of the isolates in each species in correlation with epidemiological mapping of pasteurellosis in Sharkia Governorate, Egypt. The obtained data revealed a total occurrence of 9.4% (30/317) of P. multocida among the examined birds (chickens, ducks, quails, and turkeys). The incidence rate was influenced by avian species, climate, breed, age, clinical signs, and sample type. Antimicrobial susceptibility testing revealed that all isolates were sensitive to florfenicol and enrofloxacin, while 86.6 and 73.3% of the isolates displayed resistance to amoxicillin-clavulanic acid and erythromycin, respectively. All of the P. multocida isolates showed a multiple-drug resistant pattern with an average index of 0.43. Molecular characterization revealed that the oma87, sodA, and ptfA virulence genes were detected in the all examined P. multocida isolates. The ermX (erythromycin), blaROB-1 (β-lactam), and mcr-1(colistin) resistance genes were present in 60, 46.6, and 40% of the isolates, respectively. Ducks and quails were the most virulent and harbored species of antimicrobial-resistant genes. These results were in parallel with postmortem and histopathological examinations which detected more severe interstitial pneumonia lesions in the trachea and lung, congestion, and cellular infiltration especially in ducks. Epidemiological mapping revealed that the Fakous district was the most susceptible to pasteurellosis infection. Thus, farmers are recommended to monitor their flocks for signs of respiratory disease, seek veterinary care promptly if any birds are sick, and avoid the random usage of antibiotics. In conclusion, this study presents a comprehensive picture of the risk factors in correlation to the pathognomonic characteristics of P. multocida infection in poultry sectors to help in developing more effective strategies for prevention and control.

Decoding the enigma: unveiling the molecular transmission of avian-associated tet(X4)-positive E. coli in Sichuan Province, China

Tigecycline is considered one of the "last resort antibiotics" for treating complex infections caused by multidrug-resistant (MDR) bacteria, especially for combating clinical resistant strains that produce carbapenemases. However, the tet(X4) gene, which carried by different plasmids can mediate high levels of bacterial resistance to tigecycline, was first reported in 2019. Here, we report the emergence of the plasmid-mediated tet(X4) in avian environment of Sichuan Province. A total of 21 tet(X4)-positive Escherichia coli (E. coli) strains were isolated and identified from avian samples in selected regions, with an isolation rate of 1.6% (21/1,286), and all of them were MDR strains. Multilocus Sequence Typing (MLST) method was used to classify the 21 tet(X4)-positive E. coli into the ST206, ST761, ST155, ST1638, ST542, and ST767 types, which also belong to the 3 phylogenetic subgroups A, B1, and C. Tet(X4) is located on mobile plasmids that can be efficiently and stably propagated. The results of fitness cost experiments showed that tet(X4)-positive plasmids may incur some fitness cost to host bacteria, but different tet(X4)-positive plasmids bring about differential fitness costs. Whole-genome sequencing further confirmed the tet(X4) gene can be located on IncX1-type plasmids and the core genetic structures are ISVsa3-rdmc-tet(X4) or rdmc-tet(X4)-ISVsa3, the former is a 7 copies tandem repeat structure. In this study, we isolated and identified tet(X4)-positive E. coli from the avian origin in Sichuan, analyzed the mobility of the tet(X4) by conjugational transfer and S1-PFGE, and evaluated the biological characteristics of the tet(X4)-positive plasmid using the results of conjugational frequency, plasmid stability, and fitness costs. Finally, combined with the third-generation whole-genome sequencing analysis, the molecular transmission characteristics of the tet(X4) were preliminarily clarified, providing a scientific basis for guiding veterinary clinical use in this area, as well as risk assessment and prevention of the transfer and spread of tigecycline resistant strains or genes.

Genetic identification of avian samples recovered from solar energy installations.

Renewable energy production and development will drastically affect how we meet global energy demands, while simultaneously reducing the impact of climate change. Although the possible effects of renewable energy production (mainly from solar- and wind-energy facilities) on wildlife have been explored, knowledge gaps still exist, and collecting data from wildlife remains (when negative interactions occur) at energy installations can act as a first step regarding the study of species and communities interacting with facilities. In the case of avian species, samples can be collected relatively easily (as compared to other sampling methods), but may only be able to be identified when morphological characteristics are diagnostic for a species. Therefore, many samples that appear as partial remains, or "feather spots"-known to be of avian origin but not readily assignable to species via morphology-may remain unidentified, reducing the efficiency of sample collection and the accuracy of patterns observed. To obtain data from these samples and ensure their identification and inclusion in subsequent analyses, we applied, for the first time, a DNA barcoding approach that uses mitochondrial genetic data to identify unknown avian samples collected at solar facilities to species. We also verified and compared identifications obtained by our genetic method to traditional morphological identifications using a blind test, and discuss discrepancies observed. Our results suggest that this genetic tool can be used to verify, correct, and supplement identifications made in the field and can produce data that allow accurate comparisons of avian interactions across facilities, locations, or technology types. We recommend implementing this genetic approach to ensure that unknown samples collected are efficiently identified and contribute to a better understanding of wildlife impacts at renewable energy projects.

Analysis of Retrospective Laboratory Data on the Burden of Bacterial Pathogens Isolated at the National Veterinary Research Institute Nigeria, 2018-2021.

Farm animals harbour bacterial pathogens, which are often viewed as important indicators of animal health and determinants of food safety. To better understand the prevalence and inform treatment, we audited laboratory data at the Bacteriology Laboratory of the NVRI from 2018-2021. Antibiotics were classified into seven basic classes: quinolones, tetracyclines, beta-lactams, aminoglycosides, macrolides, nitrofuran, and cephalosporins. Trends were analysed using a generalised linear model with a log link function for the Poisson distribution, comparing proportions between years with an offset to account for the variability in the total number of organisms per year. Avian (73.18%) samples were higher than any other sample. The major isolates identified were Escherichia. coli, Salmonella spp., Klebsiella spp., Staphylococcus spp., Proteus spp., and Pseudomonas spp. We found that antimicrobial resistance to baseline antibiotics increased over the years. Of particular concern was the increasing resistance of Klebsiella spp. to cephalosporins, an important second-generation antibiotic. This finding underscores the importance of farm animals as reservoirs of pathogens harbouring antimicrobial resistance. Effective biosecurity, surveillance, and frugal use of antibiotics in farms are needed because the health of humans and animals is intricately connected.

Low Incidence of Avian Predation on the Brown Marmorated Stink Bug, Halyomorpha halys (Hemiptera: Pentatomidae), in Southeastern Orchard Systems.

In many agroecosystems, brown marmorated stink bugs (Halyomorpha halys) (Hemiptera: Pentatomidae) are polyphagous pests that cause significant economic losses to numerous crops every year. Insectivorous birds may provide a means of sustainable predation of invasive pests, such as H. halys. In forest margins surrounding peach, pecan, and interplanted peach-pecan orchards, we monitored H. halys populations with pheromone-baited traps, mist-netted birds, and collected avian fecal samples for molecular gut content analysis. We screened 257 fecal samples from 19 bird species for the presence of H. halys DNA to determine whether birds provide the biological control of this pest. Overall, we found evidence that four birds from three species consumed H. halys, including Northern cardinal (Cardinalis cardinalisis), Tufted titmouse (Baeolophus bicolor), and Carolina wren (Thryothorus ludovicianus). Halyomorpha halys captured in traps increased over time but did not vary by orchard type. Although incidence of predation was low, this may be an underestimate as a result of our current avian fecal sampling methodology. Because birds are members of the broader food web, future studies are needed to understand avian ecosystem services, especially in terms of pest control, including H. halys and other pest species.

Isolation of saprophytic filamentous fungi from avian fecal samples and assessment of its predatory activity on coccidian oocysts

Fungal strains used in the biocontrol of animal gastrointestinal parasites have been mainly isolated from pasture soil, decaying organic matter, and feces from herbivores and carnivores. However, their isolation from birds and assessment of predatory activity against avian GI parasites has been scarce thus far. This research aimed to isolate filamentous fungi from avian fecal samples and evaluate their predatory activity against coccidia. A pool of 58 fecal samples from chickens, laying hens, and peacocks, previously collected between July 2020-April 2021, were used for isolation of filamentous fungi and assessment of their in vitro predatory activity against coccidian oocysts, using Water-Agar medium and coprocultures. The Willis-flotation technique was also performed to obtain concentrated suspensions of oocysts. A total of seven Mucor isolates was obtained, being the only fungal taxa identified, and all presented lytic activity against coccidia. Isolates FR3, QP2 and SJ1 had significant coccidiostatic efficacies (inhibition of sporulation) higher than 70%, while isolates FR1, QP2 and QP1 had coccidicidal efficacies (destruction of the oocysts) of 22%, 14% and 8%, respectively, after 14 days of incubation, being a gradual and time-dependent process. To our knowledge, this is the first report regarding the isolation of native predatory fungi from avian feces and demonstration of their lytic activity against coccidia.

PCR-RFLP Analysis of Insulin-Like Growth Factor 2 Gene Polymorphisms in Two Commercial Broiler Chicken Strains (Cobb 500 and Hubbard F-15) and Their Associations with Performance Traits.

The present research aimed to study the polymorphisms of the chicken insulin-like growth factor 2 (IGF2) in two commercial broiler breeds (Cobb 500 and Hubbard F-15). In total, 300 avian blood samples were obtained. The genomic DNA was isolated using a fast salt-extraction technique. Moreover, polymerase chain reaction (PCR) was used to amplify 1146 bp fragments of the gene. The amplified fragments were subjected to restriction enzyme digestion using the HinfI endonuclease enzyme, and the digested products were separated on a 2% agarose gel. The findings indicated that there were two alleles T and C for the target locus, with frequencies of 73.3% and 26.7%, respectively. Three distinct genotype variations, TT, TC, and CC, were found, with genotype frequencies of 59.1%, 28.4%, and 12.5%, respectively. A test based on actual and anticipated frequencies of various genotypic variances of the IGF2 gene revealed that the divergence from Hardy-Weinberg equilibrium was not significant (P≤0.01) in commercial broiler breeds (Cobb 500 and Hubbard F-15) chickens. In addition, it was found that birds with genotype TC had a greater body mass at 8 weeks of age, compared to those with genotypes TT and CC. It was determined that the IGF2 gene exhibited a significant degree of variability and might be regarded as a possible genetic marker in selection and breeding programs for poultry.

Long-distance spread of Tembusu virus, and its dispersal in local mosquitoes and domestic poultry in Chongming Island, China

BackgroundChongming Island in China serves as a breeding and shelter point on the East Asian–Australasian Flyway. The resting frequency of migratory birds, abundance of mosquito populations, and the popular domestic poultry industry pose a potential risk of mosquito-borne zoonotic diseases. The aim of this study is to explore the role of migratory birds in the spread of mosquito-borne pathogens and their prevalent status on the island.MethodsWe conducted a mosquito-borne pathogen surveillance in 2021, in Chongming, Shanghai, China. Approximately 67,800 adult mosquitoes belonging to ten species were collected to investigate the presence of flaviviruses, alphaviruses, and orthobunyaviruses by RT-PCR. Genetic and phylogenetic analyses were conducted to explore the virus genotype and potential nature source. Serological survey was performed by ELISA to characterize Tembusu virus (TMUV) infection among domestic poultry.ResultsTwo strains of TMUV and Chaoyang virus (CHAOV) and 47 strains of Quang Binh virus (QBV) were detected in 412 mosquito pools, with the infection rate of 0.16, 0.16, and 3.92 per 1000 Culex tritaeniorhynchus, respectively. Furthermore, TMUVs viral RNA was found in serum samples of domestic chickens and faecal samples of migratory birds. Antibodies against TMUV were detected in domestic avian serum samples, generally ranging from 44.07% in pigeons to 55.71% in ducks. Phylogenetic analyses indicated that the TMUV detected in Chongming belonged to Cluster 3, Southeast Asia origin, and most closely related to the CTLN strain, which caused a TMUV outbreak in chickens in Guangdong Province in 2020, but distant from strains obtained previously in Shanghai, which were involved in the 2010 TMUV outbreak in China.ConclusionsWe speculate that the TMUV was imported to Chongming Island through long-distance spreading by migratory birds from Southeast Asia, followed by spill over and transmission in mosquitoes and domestic avian species, threatening the local domestic poultry. In addition, the expansion and prevalence of insect-specific flaviviruses and its simultaneous circulation with mosquito-borne virus are worthy of close attention and further study.

Membrane Rigidity‐Tunable Fusogenic Nanosensor for High Throughput Detection of Fusion‐Competent Influenza A Virus

AbstractThe emergence of fatal viruses that pose continuous threats to global health has fueled the intense effort to develop direct, accurate, and high‐throughput virus detection platforms. Current diagnostic methods, including qPCR and rapid antigen tests, indicate how much of the virus is present, whether small fragments or whole viruses. However, these methods do not indicate the probability of the virus to be active, capable of interacting with host cells and initiating the infection cycle. Herein, a sialic acid‐presenting fusogenic liposome (sLipo–Chol) nanosensor with purposefully modulated membrane rigidity to rapidly detect the fusion‐competent influenza A virus (IAV) is developed. This nanosensor possesses virus‐specific features, including hemagglutinin (HA) binding and HA‐mediated membrane fusion. It is explored how the fusogenic capability of sLipo–Chol with different membrane rigidities impacts their sensing performance by integrating Förster resonance energy transfer (FRET) pairs into the bilayers. The addition of an intact virus led to instant FRET signal changes, thus enabling the direct detection of diverse IAV subtypes—even in avian fecal samples—within an hour at room temperature. Therefore, the sensing approach, with an understanding of the cellular pathogenesis of influenza viruses, will aid in developing bioinspired nanomaterials for evolution into nanosystems to detect infection‐competent viruses.

Effect of RNA preservation methods on RNA quantity and quality of field-collected avian whole blood

The transcriptome comprises all RNA molecules in a sample, tissue, or organism. A limitation of comparative transcriptomic studies which compare gene expression between individuals often under some differing exposure or treatment, of wild avian populations continues to be sample preservation of high-quality RNA (i.e., ribonucleic acids that transfer, translate, and regulate the genetic code from DNA into proteins). Field sampling of wild bird blood provides challenges as RNA degradation progresses quickly, due in large part to the high nuclease content of avian blood and because cryopreservation is often not feasible at remote locations. The introduction of commercial buffers for preservation of RNA enables field-collected studies as these buffers deactivate nucleases which degrade target nucleic acids. We seek to compare the effectiveness of widely available RNA stabilizing buffers, RNAlater (Ambion) and DNA/RNA Shield (Zymo) at varying concentrations along with a dry ice-based flash freezing method to determine optimal preservation methods for field-collected avian blood samples. To determine optimal preservation methods, we assessed RNA quantity and quality metrics: RNA integrity numbers (RINe), rRNA ratios, and total RNA concentration. Nucleated red blood cells, a characteristic common across non-mammalian vertebrates, provide sufficient transcriptionally active material enabling the identification of potentially active gene regions from small and non-lethal samples (∼ 20 μl). A protocol was also optimized for total RNA extraction from avian blood samples with small starting volumes enabling sampling of birds with a minimum threshold of 5 g body mass. We found that RNA preservation buffers, RNAlater and DNA/RNA Shield at all concentrations provide sample protection from RNA degradation. We recommend that caution be exercised when using dry ice-based flash-freezing alone for sample preservation as these samples resulted in lower quality measures then samples in preservation buffer. Total RNA concentration was generally not affected by preservation treatment and may vary due to differences in initial sample volumes.