Genetic Characterization of Highly Pathogenic Avian Influenza A(H5N8) Virus in Pakistani Live Bird Markets Reveals Rapid Diversification of Clade 2.3.4.4b Viruses.

The highly pathogenic (HPAI) avian influenza A(H5N1) viruses have undergone reassortment with multiple non-N1-subtype neuraminidase genes since 2008, leading to the emergence of H5Nx viruses. H5Nx viruses established themselves quickly in birds and disseminated from China to Africa, the Middle East, Europe and North America. Multiple genetic clades have successively evolved through frequent mutations and reassortment, posing a continuous threat to domestic poultry and causing substantial economic losses. Live bird markets are recognized as major sources of avian-to-human infection and for the emergence of zoonotic influenza. In Pakistan, the A(H5N1) virus was first reported in domestic birds in 2007; however, avian influenza surveillance is limited and there is a lack of knowledge on the evolution and transmission of the A(H5) virus in the country. We collected oropharyngeal swabs from domestic poultry and environmental samples from six different live bird markets during 2018-2019. We detected and sequenced HPAI A(H5N8) viruses from two chickens, one quail and one environmental sample in two markets. Temporal phylogenetics indicated that all novel HPAI A(H5N8) viruses belonged to clade 2.3.4.4b, with all eight genes of Pakistan A(H5N8) viruses most closely related to 2017 Saudi Arabia A(H5N8) viruses, which were likely introduced via cross-border transmission from neighboring regions approximately three months prior to virus detection into domestic poultry. Our data further revealed that clade 2.3.4.4b viruses underwent rapid lineage expansion in 2017 and acquired significant amino acid mutations, including mutations associated with increased haemagglutinin affinity to human α-2,6 receptors, prior to the first human A(H5N8) infection in Russian poultry workers in 2020. These results highlight the need for systematic avian influenza surveillance in live bird markets in Pakistan to monitor for potential A(H5Nx) variants that may arise from poultry populations.

Demographic Characteristics and Husbandry and Biosecurity Practices of Small Poultry Flocks in Ontario, Canada.

As part of a 2 yr disease surveillance project of small poultry flocks, owners of birds submitted for postmortem examination to the Animal Health Laboratory were asked to complete a questionnaire designed to gather information on the characteristics of the flock and its environment, how the flock was managed, and biosecurity measures used. A total of 153 unique questionnaires were received. Personal consumption of meat or eggs was the most common reason for owning a small flock (69.3%). Almost all owners (97.4%) reported having chickens on their property, while 21.6% had waterfowl, 15.7% had turkeys, and 15.7% had game birds. Nearly 70% (69.9%) of the flocks had some degree of outdoor access. For those with indoor access, the most common bedding material provided was soft wood shavings (70.2%). Kitchen waste or leftovers were offered to 65.3% of flocks, and well water was the most common source of drinking water (80.6%). For flocks with indoor access, dedicated shoes and clothes were used when entering or cleaning the coop by fewer than half of owners, and shoes were rarely disinfected before or after contact with the flock. Most owners (93.8%) reported washing their hands after contact with their birds, although only 48.3% reported washing their hands before contact. Among owners who sourced birds from a hatchery, only 36.8% indicated that the birds had been vaccinated, and 21.1% were unsure if vaccines had been administered. Among owners using medication (60.5%), the use of antibiotics was common (60.9%). Overall, questionnaire responses describe a wide range of husbandry and biosecurity practices, often suboptimal, and point out the need for educational material for Ontario small flock owners.

Artículo Regular­Características demográficas y prácticas de cría y bioseguridad de pequeñas parvadas de aves de corral en Ontario, Canadá. Como parte de un proyecto de vigilancia de enfermedades de dos años de pequeñas parvadas avícolas, se solicitó a los propietarios de aves remitidaspara exámenes post mortem en el Laboratorio de Sanidad Animal que completaran un cuestionario diseñado para recopilar información sobre las características de la parvada y su entorno, cómo se manejó la parva y se que medidas de bioseguridad fueron usadas. Se recibieron un total de 153 cuestionarios únicos. El consumo personal de carne o huevo fue la razón más común para tener una parvada pequeña (69.3%). Casi todos los propietarios (97.4%) informaron tener pollos en su propiedad, mientras que el 21.6% tenía aves acuáticas, el 15.7% tenía pavos y el 15.7% tenía aves para caza. Casi el 70% (69.9%) de las parvadas tenían algún grado de acceso al aire libre. Para aquellos con acceso interior, el material de cama más común proporcionado fue viruta de madera blanda (70.2%). Al 65.3% de las parvadas se le ofrecieron desperdicios de cocina o sobras y el agua de pozo fue la fuente más común de agua potable (80.6%). En el caso de las parvadas con acceso al interior, menos de la mitad de los propietarios utilizaron zapatos y ropa especiales para entrar o limpiar los alojamientos y los zapatos rara vez se desinfectaban antes o después del contacto con la parvada. La mayoría de los propietarios (93.8%) informaron lavarse las manos después del contacto con sus aves, aunque solo el 48.3% informó lavarse las manos antes del contacto. Entre los propietarios que obtuvieron aves de una planta incubadora, solo el 36.8% indicó que las aves habían sido vacunadas y el 21.1% no estaba seguro de si se habían administrado vacunas. Entre los propietarios que usaban medicación (60.5%), el uso de antibióticos era común (60.9%). En general, las respuestas al cuestionario describen una amplia gama de prácticas de críanza y bioseguridad, a menudo subóptimas, y señalan la necesidad de material educativo para los propietarios de pequeñas parvadas en Ontario.

Geometrical characteristics of eggs from 3 poultry species.

We studied the correlations between egg geometrical parameters (i.e., egg shape index, sphericity, geometric mean diameter, surface area, and volume) and eggshell qualities, or the organic matrix in eggshell. Eggs were collected from 5 poultry breeds belonging to 3 species (commercial Hy-line Brown Chicken, Shaoxing Duck, Jinding Duck, Taihu Goose, and Zhedong White Goose). The geometrical parameters showed high variation among 3 species of poultry, and even between breeds in the same species. The five geometrical parameters were grouped into 2 sets, one contained shape index and sphericity, the other comprised geometric mean diameter, surface area, and volume. The parameters in the same set can be perfectly fitted to one another. Egg weight, shell membrane weight, and calcified shell weight were significantly correlated with geometric mean diameter, surface area, and volume. In accordance with false discovery rate-adjusted P value, both shell membrane relative weight and calcified shell thickness showed no significant correlations with any of the geometrical parameters. However, the correlations between geometrical parameters and other shell variables (calcified shell weight, shell relative weight, calcified shell thickness uniformity, and eggshell breaking strength) depend on breed. Both constitutive proportions and percentage contents of 3 eggshell matrix components (acid-insoluble, water-insoluble, and both acid and water facultative-soluble matrix) had no effects on egg shape and size. The correlations between the amounts of various shell matrix, egg shape and size depend on breed or species. This study provides a methodology and the correlation between geometrical parameters and eggshell qualities, and between geometrical parameters and organic matrix components in calcified shells.

Development of a DNA metabarcoding method for the identification of fifteen mammalian and six poultry species in food.

Meat products are prone to adulteration by the replacement of meat from more expensive animal species with meat from cheaper sources. We present a DNA metabarcoding method allowing the identification and differentiation of 15 mammalian and six poultry species in foodstuffs. The method, developed on the MiSeq® platform, targets a mitochondrial 16S rDNA region recently found to be suitable for the differentiation of 300 mammalian species. We designed a novel primer pair for poultry and applied it in combination with the primer pair for mammalian species in a duplex assay. The applicability of the method was investigated by analysing DNA extracts from muscle, DNA extract mixtures and extracts from model sausages. Our results indicated that the species of interest can be identified, differentiated and detected down to a proportion of 0.1%. Since 96 samples can be sequenced in one run, the method has high potential for application in routine analysis.

Global trends in antimicrobial use in food animals.

Demand for animal protein for human consumption is rising globally at an unprecedented rate. Modern animal production practices are associated with regular use of antimicrobials, potentially increasing selection pressure on bacteria to become resistant. Despite the significant potential consequences for antimicrobial resistance, there has been no quantitative measurement of global antimicrobial consumption by livestock. We address this gap by using Bayesian statistical models combining maps of livestock densities, economic projections of demand for meat products, and current estimates of antimicrobial consumption in high-income countries to map antimicrobial use in food animals for 2010 and 2030. We estimate that the global average annual consumption of antimicrobials per kilogram of animal produced was 45 mg⋅kg(-1), 148 mg⋅kg(-1), and 172 mg⋅kg(-1) for cattle, chicken, and pigs, respectively. Starting from this baseline, we estimate that between 2010 and 2030, the global consumption of antimicrobials will increase by 67%, from 63,151 ± 1,560 tons to 105,596 ± 3,605 tons. Up to a third of the increase in consumption in livestock between 2010 and 2030 is imputable to shifting production practices in middle-income countries where extensive farming systems will be replaced by large-scale intensive farming operations that routinely use antimicrobials in subtherapeutic doses. For Brazil, Russia, India, China, and South Africa, the increase in antimicrobial consumption will be 99%, up to seven times the projected population growth in this group of countries. Better understanding of the consequences of the uninhibited growth in veterinary antimicrobial consumption is needed to assess its potential effects on animal and human health.

Species identification of ten common farm animals based on mitochondrial 12S rRNA gene polymorphisms.

Bio-techniques such as genetic manipulation, marker-assisted selection, and identity test have largely facilitated the modern animal production practices. In the present study, we established a reliable and cost-effective molecular method of species identification for common farm animals. We first (re-)analyzed 179 mitochondrial 12S rRNA gene sequences of ten farm animal species to determine the intra-species and species-specific variations. The PCR-RFLP method was subsequently designed to identify these species by using endonucleases BshNI, ScaI, AluI, and BfaI. The poultry and livestock species were first discriminated by one double-digestion of both BshNI and ScaI, which generated different fragment patterns (325 bp and 115 bp for poultry vs. 364 bp and 76 bp for livestock). The ten species could be further discerned according to species-specific restriction pattern by subjecting to digestion of AluI and BfaI, respectively. Our approach would be more reliable by taking the intra-species variations into consideration and could be applied to species identity test, commercial fraud, and wildlife crime.

Evolution of highly pathogenic avian influenza H5N1 viruses in Egypt indicating progressive adaptation.

Highly pathogenic avian influenza (HPAI) virus of the H5N1 subtype was first diagnosed in poultry in Egypt in 2006, and since then the disease became enzootic in poultry throughout the country, affecting the poultry industry and village poultry as well as infecting humans. Vaccination has been used as a part of the control strategy to help to control the disease. Epidemiological data with sequence analysis of H5N1 viruses is important to link the mechanism of virus evolution in Egypt. This study describes the evolutionary pattern of Egyptian H5N1 viruses based on molecular characterization for the isolates collected from commercial poultry farms and village poultry from 2006 to 2011. Genetic analysis of the hemagglutinin (HA) gene was done by sequencing of the full-length H5 gene. The epidemiological pattern of disease outbreaks in Egyptian poultry farms seems to be seasonal with no specific geographic distribution across the country. The molecular epidemiological data revealed that there are two major groups of viruses: the classic group of subclade 2.2.1 and a variant group of 2.2.1.1. The classic group is prevailing mainly in village poultry and had fewer mutations compared to the originally introduced virus in 2006. Since 2009, this group has started to be transmitted back to commercial sectors. The variant group emerged by late 2007, was prevalent mainly in vaccinated commercial poultry, mutated continuously at a higher rate until 2010, and started to decline in 2011. Genetic analysis of the neuraminidase (NA) gene and the other six internal genes indicates a grouping of the Egyptian viruses similar to that obtained using the HA gene, with no obvious reassortments. The results of this study indicate that HPAI-H5N1 viruses are progressively evolving and adapting in Egypt and continue to acquire new mutations every season.

Níveis nutricionais de cálcio para aves de corte ISA label criadas sob semiconfinamento / Dietary calcium levels for ISA label broilers raised in semi-confined system

Determinaram-se os níveis nutricionais de cálcio (Ca) para aves, machos e fêmeas, da linhagem ISA Label, nas fases inicial (um a 28 dias), crescimento (28 a 56 dias) e final (56 a 84 dias). Foram realizados três ensaios, um para cada fase, e, em cada ensaio, 480 aves com idade correspondente à fase de criação foram alojadas em 24 unidades experimentais com áreas de abrigo e de pastejo. Foi utilizado delineamento inteiramente ao acaso, em esquema fatorial 4x2 (Ca e sexo), totalizando oito tratamentos com três repetiç ões de 20 aves. Avaliaram-se: ganho de peso (GP); consumo de dieta (CD); conversão alimentar (CA); teores de fósforo (PT), de cálcio (CaT) e de cinzas na tíbia (CT) e resistência à quebra óssea (RQO). Na fase inicial, recomenda-se 1,16 por cento de Ca na dieta, para aves de ambos os sexos, na fase de crescimento, 0,78 e 0,88 por cento de Ca para machos e fêmeas, respectivamente, e, na fase final, 0,69 por cento de Ca na dieta para ambos os sexos.

The nutritional calcium (Ca) levels were determined for ISA Label broilers in the starter (1 to 28 days), growing (28 to 56 days), and finishing (56 to 84 days) phases. Three trials were conducted, one for each phase and in each trial, 480 birds with age corresponding to the phase were housed in 24 experimental units with shelter and pasture areas. The experimental design was completely randomized in a factorial arrangement of 4x2 (Ca levels and sex), totaling eight treatments with three replicates of 20 birds. Body weight gain (BWG); feed intake (FI); feed per gain (FG); contents of phosphorus (TP), calcium (TCa) and ash (TA) in tibia; and bone breaking strength (BSB) were evaluated. At the starter phase, 1.16 percent of Ca is recommended in the diet for birds of both sexes. At the growing phase, 0.78 and 0.88 percent of Ca for males and females, respectively, are recommended and at the finishing phase, the level of 0.69 percent of Ca is recommended in diet for both sexes.

Agro-ecological features of the introduction and spread of the highly pathogenic avian influenza (HPAI) H5N1 in northern Nigeria.

Nigeria was the first African country to report highly pathogenic avian influenza (HPAI) H5N1 virus outbreaks in February 2006 and has since been the most severely hit country in sub-Saharan Africa. A retrospective survey carried out towards the end of 2007, coupled with follow-up spatial analysis, support the notion that the H5N1 virus may have spread from rural areas of northern Nigeria near wetlands frequented by palaearctic migratory birds. Possibly, this could have happened already during November to December 2005, one or two months prior to the first officially reported outbreak in a commercial poultry farm (Kaduna state). It is plausible that backyard poultry played a more important role in the H5N1 propagation than thought previously. Farming landscapes with significant numbers of domestic ducks may have helped to bridge the geographical and ecological gap between the waterfowl in the wetlands and the densely populated poultry rich states in north-central Nigeria, where the virus had more sizeable, visible impact.

Analysis of genetic diversity and phylogenetic relationships among red jungle fowls and Chinese domestic fowls.

Genetic diversity and phylogenetic relationships among 568 individuals of two red jungle fowl subspecies (Gallus gallus spadiceus in China and Gallus gallus gallus in Thailand) and 14 Chinese domestic chicken breeds were evaluated with 29 microstaellite loci, the genetic variability within population and genetic differentiation among population were estimated, and then genetic diversity and phylogenetic relationships were analyzed among red jungle fowls and Chinese domestic fowls. A total of 286 alleles were detected in 16 population with 29 microsatellite markers and the average number of the alleles observed in 29 microsatellite loci was 9.86+/-6.36. The overall expected heterozygosity of all population was 0.6708+/-0.0251, and the number of population deviated from Hardy-Weinberg equilibrium per locus ranged from 0 to 7. In the whole population, the average of genetic differentiation among population, measured as FST value, was 16.7% (P<0.001), and all loci contributed significantly (P<0.001) to this differentiation. It can also be seen that the deficit of heterozygotes was very high (0.015) (P<0.01). Reynolds' distance values varied between 0.036 (Xiaoshan chicken-Luyuan chicken pair) and 0.330 (G. gallus gallus-Gushi chicken pair). The Nm value ranged from 0.533 (between G. gallus gallus and Gushi chicken) to 5.833 (between Xiaoshan chicken and Luyuan chicken). An unrooted consensus tree was constructed using the neighbour-joining method and the Reynolds' genetic distance. The heavy-body sized chicken breeds, Luyuan chicken, Xiaoshan chicken, Beijing Fatty chicken, Henan Game chicken, Huainan Partridge and Langshan chicken formed one branch, and it had a close genetic relationship between Xiaoshan chicken-Luyuan chicken pair and Chahua chicken-Tibetan chicken pair. Chahua chicken and Tibetan chicken had closer genetic relationship with these two subspecies of red jungle fowl than other domestic chicken breeds. G. gallus spadiceus showed closer phylogenetic relationship with Chinese domestic chicken breeds than G. gallus gallus. All 29 microstaellite loci in this study showed high levels of polymorphism and significant genetic differentiation was observed among two subspecies of red jungle fowl and 14 Chinese domestic chicken breeds. The evolutional dendrogram is as follows: evolutional breeds-->primitive breeds (Chahua chicken and Tibetan)-->red jungle fowl in China (G. gallus spadiceus)-->red jungle fowl in Thailand (G. gallus gallus). The results supported the theory that the domestic fowls might originate from different subspecies of red jungle fowl and Chinese domestic fowls had independent origin.

Multiple maternal origins of chickens: out of the Asian jungles.

Domestic chickens have long been important to human societies for food, religion, entertainment, and decorative uses, yet the origins and phylogeography of chickens through Eurasia remain uncertain. Here, we assessed their origins and phylogeographic history by analyzing the mitochondrial DNA hypervariable segment I (HVS-I) for 834 domestic chickens (Gallus gallus domesticus) across Eurasia as well as 66 wild red jungle fowls (Gallus gallus) from Southeast Asia and China. Phylogenetic analyses revealed nine highly divergent mtDNA clades (A-I) in which seven clades contained both the red jungle fowls and domestic chickens. There was no breed-specific clade in the chickens. The clades A, B, and E are distributed ubiquitously in Eurasia, while the other clades were restricted to South and Southeast Asia. Clade C was mainly distributed in Japan and Southeast China, while clades F and G were exclusive to Yunnan, China. The geographic distribution of clade D was closely related to the distribution of the pastime of cock fighting. Statistical tests detect population expansion within each subclade. These distinct distribution patterns and expansion signatures suggest that different clades may originate from different regions, such as Yunnan, South and Southwest China and/or surrounding areas (i.e., Vietnam, Burma, and Thailand), and the Indian subcontinent, respectively, which support the theory of multiple origins in South and Southeast Asia.

Update on molecular epidemiology of H1, H5, and H7 influenza virus infections in poultry in North America.

Avian influenza is endemic in wild birds in North America, and the virus routinely has been transmitted from this reservoir to poultry. Influenza, once introduced into poultry, can become endemic within the poultry population. It may be successfully eradicated by human intervention, or the virus may fail to successfully spread on its own. In the last 5 yr, influenza virus has been isolated from poultry in the United States on numerous occasions, and, with the use of molecular epidemiology, the relationships of these different viruses can be determined. There are 15 different hemagglutinin subtypes of avian influenza viruses, but infections with virus of H5 and H7 subtypes are of the most concern because of the potential for these viruses to mutate to the highly pathogenic form of the virus. Most of the influenza isolations in the United States have been associated with the live-bird markets (LBMs) in the Northeast. This has included primarily H7N2 influenza viruses, but also H7N3, H5N2, and other subtypes. Most of the H7N2 viruses were part of a single lineage that was first observed in 1994, but new introductions of H7N2 and H7N3 were also observed. The predominant H7N2 LBM lineage of virus spread to large commercial poultry operations on at least three occasions since 1997, with the largest outbreak occurring in Virginia in 2002. The H5N2 viruses in the LBMs included viruses from domestic ducks, gamebirds, and environmental samples. Some H5N2 viruses isolated in different years and in different locations had a high degree of sequence relatedness, although the reservoir source, if it is endemic, has not been identified. Finally, an H1N2 virus, associated with a drop in egg production, was isolated from turkeys in Missouri in 1999. This virus was a complex reassortant with swine, human, and avian influenza genes that was similar to recent swine isolates from the Midwest. Additional serologic evidence suggests that flocks in other states were infected with a H1N2 virus.

Expressed sequence tags for the chicken genome from a normalized, ten-day-old white leghorn whole embryo cDNA library. 2. Comparative DNA sequence analysis of guinea fowl, quail, and turkey genomes.

Accelerated efforts to develop a high-utility chicken genome map have resulted in the development of resources that may be useful for genetic analysis in other economically important poultry species. Here we describe a total of 26 comparative genomic DNA sequences (CGS) for the guinea fowl, Japanese quail, and domestic turkey developed using 10 primer pairs specific for 10 previously reported, unique, chicken expressed sequence tags (EST). The total length of CGS developed for each of the three species was 4,193, 4,597, and 6,057 bp in quail, turkey, and guinea fowl, respectively. About 70% of the CGS showed significant sequence similarity to reference database sequences, including the reference chicken EST and other avian and nonavian genes. A majority of the between-species comparisons of the CGS from all but two primer pairs were significant and ranged from 81 to 99%. The percentage similarity of the CGS appears to be a function of phylogenetic relatedness and was generally higher for comparisons between the chicken, quail, and turkey and lower between the guinea fowl and chicken, quail, or turkey. Maximum likelihood estimation of the phylogenetic relationships using CGS from two primer pairs also showed a closer relationship, as expected, among chicken, quail, and turkey than between guinea fowl and either chicken, quail, or turkey. Within the guinea fowl, quail, and turkey CGS developed, the total number of single nucleotide polymorphisms detected was 28, 17, and 14, respectively. Together, these resources represent tools that will facilitate genetic analysis of species that have been studied very little and our understanding of their genomes and genome evolution.

Cambios en el contenido de tocoferoles de la yema de huevo en función del tiempo y del tipo de almacenamiento / Changes in the content of tocopherols in yolk acoording to time and type of storage

En este estudio se ha determinado como cambia el contenido de vitamina E (a-tocoferol, b-tocoferol, c-tocoferol. d-tocoferol) y acetato de a-tocoferol de la yema de huevo durante su almacenamiento. Éste se realizó bajo dos formas distintas de conservación (temperatura ambiente a 18-23ºC y refrigeración a 4ºC). Los resultados obtenidos muestran que el contenido de acetato de a-tocoferol disminuye significativamente en ambos tipos de conservación (F = 7,33; P<= 0,001) durante el alamcenamiento. Sin embargo, el contenido de a, b+c y d-tocoferoles no cambia significativamente. Los niveles de a-tocoferol, b-tocoferol y acetato de a-tocoferol fueron significativamente mayores en las yemas de los huevos refrigerados que en las de los huevos almacenados a temperatura ambiente (F = 21,70 y P<=0,001; F =7,27 y P<=0,01; F =7,90 y P<=0,01, respectivamente). Por el contrario, el nivel de b+c-tocoferoles no presenta diferencias significativas en función del tipo de conservación. (AU)