The small hydrophobic (SH) gene of North American turkey AMPV-C does not attenuate nor modify host tropism in recombinant European duck AMPV-C.

Subgroup C Avian Metapneumoviruses (AMPV-C) has two lineages, one mostly in turkeys and one mostly in ducks. To investigate the molecular basis of AMPV-C host tropism, a reverse genetics system for a duck AMPV-C virus was developed. A recombinant copy and a recombinant virus in which the SH protein had been exchanged for that of a turkey AMPV-C were rescued. No change in cytopathogenic effect or replication profile in vitro were observed for either virus compared to the wild type. In SPF Muscovy ducks the wild type and its recombinant copy were equally pathogenic. Exchanging the SH in the recombinant copy produced the same results. In SPF turkeys, neither recombinant virus was pathogenic, although both showed a low level of replication. Thus, from the current model, it appears that AMPV-C SH proteins derived from the different species are compatible and that turkey SH does not affect duck AMPV-C pathogenicity.

Emergence and multiple reassortments of French 2015-2016 highly pathogenic H5 avian influenza viruses.

From November 2015 to August 2016, 81 outbreaks of highly pathogenic (HP) H5 avian influenza virus were detected in poultry farms from South-Western France. These viruses were mainly detected in farms raising waterfowl, but also in chicken or guinea fowl flocks, and did not induce severe signs in waterfowl although they did meet the HP criteria. Three different types of neuraminidases (N1, N2 and N9) were associated with the HP H5 gene. Full genomes sequences of 24 H5HP and 6 LP viruses that circulated in the same period were obtained by next generation sequencing, from direct field samples or after virus isolation in SPF embryonated eggs. Phylogenetic analyses of the eight viral segments confirmed that they were all related to the avian Eurasian lineage. In addition, analyses of the "Time of the Most Recent Common Ancestor" showed that the common ancestor of the H5HP sequences from South-Western France could date back to early 2014 (±1 year). This pre-dated the first detection of H5 HP in poultry farms and was consistent with a silent circulation of these viruses for several months. Finally, the phylogenetic study of the different segments showed that several phylogenetic groups could be established. Twelve genotypes of H5HP were detected implying that at least eleven reassortment events did occur after the H5HP cleavage site emerged. This indicates that a large number of co-infections with both highly pathogenic H5 and other avian influenza viruses must have occurred, a finding that lends further support to prolonged silent circulation.

Effect of a photoperiodic green light programme during incubation on embryo development and hatch process.

This study was conducted to evaluate the effect of a 12-h light, 12-h dark (12L : 12D) photoperiod of green light during day 1 to day 18 of incubation time, on embryo growth, hormone concentration and the hatch process. In the test group, monochromatic light was provided by a total of 204 green light-emitting diodes (522 nm) mounted in a frame which was placed above the top tray of eggs to give even spread of illumination. No light-dark cycle was used in the control group. Four batches of eggs (n=300/group per batch) from fertile Ross 308 broiler breeders were used in this experiment. The beak length and crown-rump length of embryos incubated under green light were significantly longer than that of control embryos at day 10 and day 12, respectively (P<0.01). Furthermore, green light-exposed embryos had a longer third toe length compared with control embryos at day 10, day 14 and day 17 (P=0.02). At group level (n=4 batches), light stimulation had no effect on chick weight and quality at take-off, the initiation of hatch and hatch window. However, the individual hatching time of the light exposure focal chicks (n=33) was 3.4 h earlier (P=0.49) than the control focal chicks (n=36) probably due to the change in melatonin rhythm of the light group. The results of this study indicate that green light accelerates embryo development and alters hatch-related hormones (thyroid and corticosterone), which may result in earlier hatching.

Single reaction, real time RT-PCR detection of all known avian and human metapneumoviruses.

Current molecular methods for the detection of avian and human metapneumovirus (AMPV, HMPV) are specifically targeted towards each virus species or individual subgroups of these. Here a broad range SYBR Green I real time RT-PCR was developed which amplified a highly conserved fragment of sequence in the N open reading frame. This method was sufficiently efficient and specific in detecting all MPVs. Its validation according to the NF U47-600 norm for the four AMPV subgroups estimated low limits of detection between 1000 and 10copies/µL, similar with detection levels described previously for real time RT-PCRs targeting specific subgroups. RNA viruses present a challenge for the design of durable molecular diagnostic test due to the rate of change in their genome sequences which can vary substantially in different areas and over time. The fact that the regions of sequence for primer hybridization in the described method have remained sufficiently conserved since the AMPV and HMPV diverged, should give the best chance of continued detection of current subgroups and of potential unknown or future emerging MPV strains.

First complete genome sequence of European turkey coronavirus suggests complex recombination history related with US turkey and guinea fowl coronaviruses.

A full-length genome sequence of 27,739  nt was determined for the only known European turkey coronavirus (TCoV) isolate. In general, the order, number and size of ORFs were consistent with other gammacoronaviruses. Three points of recombination were predicted, one towards the end of 1a, a second in 1b just upstream of S and a third in 3b. Phylogenetic analysis of the four regions defined by these three points supported the previous notion that European and American viruses do indeed have different evolutionary pathways. Very close relationships were revealed between the European TCoV and the European guinea fowl coronavirus in all regions except one, and both were shown to be closely related to the European infectious bronchitis virus (IBV) Italy 2005. None of these regions of sequence grouped European and American TCoVs. The region of sequence containing the S gene was unique in grouping all turkey and guinea fowl coronaviruses together, separating them from IBVs. Interestingly the French guinea fowl virus was more closely related to the North American viruses. These data demonstrate that European turkey and guinea fowl coronaviruses share a common genetic backbone (most likely an ancestor of IBV Italy 2005) and suggest that this recombined in two separate events with different, yet related, unknown avian coronaviruses, acquiring their S-3a genes. The data also showed that the North American viruses do not share a common backbone with European turkey and guinea fowl viruses; however, they do share similar S-3a genes with guinea fowl virus.

Novel Receptor Specificity of Avian Gammacoronaviruses That Cause Enteritis.

UNLABELLED: Viruses exploit molecules on the target membrane as receptors for attachment and entry into host cells. Thus, receptor expression patterns can define viral tissue tropism and might to some extent predict the susceptibility of a host to a particular virus. Previously, others and we have shown that respiratory pathogens of the genus Gammacoronavirus, including chicken infectious bronchitis virus (IBV), require specific α2,3-linked sialylated glycans for attachment and entry. Here, we studied determinants of binding of enterotropic avian gammacoronaviruses, including turkey coronavirus (TCoV), guineafowl coronavirus (GfCoV), and quail coronavirus (QCoV), which are evolutionarily distant from respiratory avian coronaviruses based on the viral attachment protein spike (S1). We profiled the binding of recombinantly expressed S1 proteins of TCoV, GfCoV, and QCoV to tissues of their respective hosts. Protein histochemistry showed that the tissue binding specificity of S1 proteins of turkey, quail, and guineafowl CoVs was limited to intestinal tissues of each particular host, in accordance with the reported pathogenicity of these viruses in vivo. Glycan array analyses revealed that, in contrast to the S1 protein of IBV, S1 proteins of enteric gammacoronaviruses recognize a unique set of nonsialylated type 2 poly-N-acetyl-lactosamines. Lectin histochemistry as well as tissue binding patterns of TCoV S1 further indicated that these complex N-glycans are prominently expressed on the intestinal tract of various avian species. In conclusion, our data demonstrate not only that enteric gammacoronaviruses recognize a novel glycan receptor but also that enterotropism may be correlated with the high specificity of spike proteins for such glycans expressed in the intestines of the avian host. IMPORTANCE: Avian coronaviruses are economically important viruses for the poultry industry. While infectious bronchitis virus (IBV), a respiratory pathogen of chickens, is rather well known, other viruses of the genus Gammacoronavirus, including those causing enteric disease, are hardly studied. In turkey, guineafowl, and quail, coronaviruses have been reported to be the major causative agent of enteric diseases. Specifically, turkey coronavirus outbreaks have been reported in North America, Europe, and Australia for several decades. Recently, a gammacoronavirus was isolated from guineafowl with fulminating disease. To date, it is not clear why these avian coronaviruses are enteropathogenic, whereas other closely related avian coronaviruses like IBV cause respiratory disease. A comprehensive understanding of the tropism and pathogenicity of these viruses explained by their receptor specificity and receptor expression on tissues was therefore needed. Here, we identify a novel glycan receptor for enteric avian coronaviruses, which will further support the development of vaccines.

Higher levels of CO2 during late incubation alter the hatch time of chicken embryos.

1. It has been reported that the increasing CO2 tension triggers the embryo to pip the air cell and emerge from the egg. However, the mechanism by which higher CO2 concentrations during the last few days of incubation affect chick physiology and the hatching process is unclear. This study investigated the effect of CO2 concentrations up to 1% during pipping, on the onset and length of the hatch window (HW) and chick quality. 2. Four batches of Ross 308 broiler eggs (600 eggs per batch) were incubated in two small-scale custom-built incubators (Petersime NV). During the final 3 d of incubation, control eggs were exposed to a lower CO2 concentration (0.3%), while the test eggs experienced a higher CO2 concentration programme (peak of 1%). 3. There were no significant differences in blood values, organ weight and body weight. There was also no difference in hatchability between control and test groups. However, a small increase in the chick weight and the percentage of first class chicks was found in the test groups. Furthermore, plasma corticosterone profiles during hatching were altered in embryos exposed to higher CO2; however, they dropped to normal levels at d 21 of incubation. Importantly, the hatching process was delayed and synchronised in the test group, resulting in a narrowed HW which was 2.7 h shorter and 5.3 h later than the control group. 4. These results showed that exposing chicks to 1% CO2 concentration during pipping did not have negative impacts on physiological status of newly hatched chicks. In addition, it may have a significant impact on the physiological mechanisms controlling hatching and have benefits for the health and welfare of chickens by reducing the waiting time after hatching.

An insight into the heat and mass transfer mechanisms of eggshells hatching broiler chicks and its effects to the hatcher environment.

Thermodynamic study of incubated eggs is an important component in the optimisation of incubation processes. However, research on the interaction of heat and moisture transfer mechanisms in eggs is rather limited and does not focus on the hatching stage of incubation. During hatch, both the recently hatched chick and the broken eggshell add extra heat and moisture contents to the hatcher environment. In this study, we have proposed a novel way to estimate thermodynamically the amount of water evaporated from a broken eggshell during hatch. The hypothesis of this study considers that previously reported drops in eggshell temperature during hatching of chicks is the result remaining water content evaporating from the eggshell, released on the inner membrane by the recently hatched wet chick, just before hatch. To reproduce this process, water was sprayed on eggshells to mimic the water-fluid from the wet body of a chick. For each sample of eggshell, the shell geometry and weight, surface area and eggshell temperature were measured. Water evaporation losses and convection coefficient were calculated using a novel model approach considering the simultaneous heat and mass transfer profiles in an eggshell. The calculated average convective coefficient was 23.9 ± 7.5 W/m(2) °C, similar to previously reported coefficients in literature as a function of 0.5-1m/s air speed range. Comparison between measured and calculated values for the water evaporation showed 68% probability accuracy, associated to the use of an experimentally derived single heat transfer coefficient. The results support our proposed modelling approach of heat and mass transfer mechanisms. Furthermore, by estimating the amount of evaporated water in an eggshell post-hatch, air humidity levels inside the hatcher can be optimised to ensure wet chicks dry properly while not dehydrating early hatching chicks.

Physiological status of broiler chicks at pulling time and the relationship to duration of holding period.

Newly hatched chicks may be held longer than 48 h and experience long periods of fasting in commercial hatcheries. Limited information is known about the physiological status of chicks in such situations, due to the difficulty of precisely recording time of hatch. This study investigated the effect of the time from hatch to pulling (holding period) on physiological measures/parameters in 109 broiler chicks. Fertile Ross 308 eggs were incubated in a custom built small-scale incubator. The individual hatching time of each focal chick was determined using eggshell temperature monitoring. At 'pulling' (512 h of incubation time), the quality of focal chicks was assessed using the chick scoring method and physiological parameters were measured including BW, organ (heart, liver and stomach) weights, blood values and plasma corticosterone level. The time from hatch to pulling varied from 7.58 to 44.97 h. Egg weight at setting was significantly correlated with chick BW and weight of organs at pulling, but had no effect on chick quality, blood values and plasma corticosterone. Relative BW at pulling was negatively associated with the duration of holding period (P=0.002). However, there was a positive correlation between relative stomach weight and the duration of the holding period (P<0.001). As the holding period duration increased, there was a trend that blood partial pressure of oxygen, haematocrit and haemoglobin also increased, and blood partial pressure of carbon dioxide, total carbon dioxide and bicarbonate decreased (P<0.05). A wide range of plasma corticosterone was observed from chicks that had experienced different durations of holding period. We conclude that shortening the hatch window and minimising the number of chicks that experience a long holding period before pulling may improve chick quality and physiological status, which may be due to unfavourable environmental conditions that include feed and water deprivation.

Effect of species-specific sound stimulation on the development and hatching of broiler chicks.

1. Previous research has reported that chicken embryos develop a functionary auditory system during incubation and that prenatal sound may play an important role in embryo development and alter the hatch time. In this study the effects of prenatal auditory stimulation on hatch process, hatch performance, the development of embryo and blood parameters were investigated. 2. Four batches of Ross 308 broiler breeder eggs were incubated either in control or in sound-stimulated groups. The sound-stimulated embryos were exposed to a discontinuous sound of species-specific calls by means of a speaker at 72 dB for 16 h a day: maternal calls from d 10 to d 19 of incubation time and embryo/chick calls from d 19 until hatching. The species-specific sound was excluded from the control group. 3. The onset of hatch was delayed in the sound-stimulated group compared to the controls. This was also supported by comparison of the exact hatching time of individual focal chicks within the two groups. However, the sound-stimulated embryos had a lower hatchability than the control group, mainly due to significantly increased numbers of late deaths. 4. The embryos exhibited a similar growth pattern between the sound-stimulated group and the control group. Although sound exposure decreased body weight at d 16, no consistent effect of sound on body weight at incubation stage was observed. Species-specific sound stimulation also had no impact on chick quality, blood values and plasma corticosterone concentrations during hatch.

Effect of transportation duration of 1-day-old chicks on postplacement production performances and pododermatitis of broilers up to slaughter age.

This experiment studied the effect of transportation duration of 1-d-old chicks on dehydration, mortality, production performance, and pododermatitis during the growout period. Eggs from the same breeder flock (Ross PM3) were collected at 35, 45, and 56 wk of age, for 3 successive identical experiments. In each experiment, newly hatched chicks received 1 of 3 transportation duration treatments from the hatchery before placement in the on-site rearing facility: no transportation corresponding to direct placement in less than 5 min (T00), or 4 (T04) or 10 h (T10) of transportation. The chicks were housed in 35-m(2) pens (650 birds each) and reared until 35 d old. Hematocrit and chick BW were measured on sample chicks before and after transportation. During the growout period, bird weight, feed uptake, and feed conversion ratio were measured weekly until slaughter. Transportation duration affected BW; T00 groups had a significantly higher BW than T04 and T10 transported birds but this effect lasted only until d 21. No clear effect on hematocrit, feed uptake, feed conversion ratio, or mortality was observed for birds transported up to 10 h. The decrease in weight in T10 birds was associated with less severe pododermatitis. Increasing age of the breeder flock was correlated with reduced egg fertility and hatchability, and also with higher quality and BW of hatched chicks. Chicks from older breeders also exhibited reduced mortality during the growout period.

Embryonic development and the physiological factors that coordinate hatching in domestic chickens.

Embryonic growth and development is influenced by both endogenous and exogenous factors. The purpose of this review is to discuss the critical stages of chick embryonic development in relation to functional maturation of numerous organ systems, the acquisition of thermoregulation, and the hatching process. In addition, the mechanism of hatching, including sound synchronization and hormonal and environmental stimulation, will be discussed. Finally, the importance of effective hatching synchronization mechanisms will also be highlighted.

Monitoring the hatch time of individual chicken embryos.

This study investigated variations in eggshell temperature (T(egg)) during the hatching process of broiler eggs. Temperature sensors monitored embryo temperature by registering T(egg) every minute. Measurements carried out on a sample of 40 focal eggs revealed temperature drops between 2 to 6°C during the last 3 d of incubation. Video cameras recorded the hatching process and served as the gold standard reference for manually labeling the hatch times of chicks. Comparison between T(egg) drops and the hatch time of individuals revealed a time synchronization with 99% correlation coefficient and an absolute average time difference up to 25 min. Our findings suggest that attaching temperature sensors to eggshells is a precise tool for monitoring the hatch time of individual chicks. Individual hatch monitoring registers the biological age of chicks and facilitates an accurate and reliable means to count hatching results and manage the hatch window.

Transfer of melamine in some poultry products.

This study deals with the transfer of melamine from poultry feed to certain poultry products, such as eggs and meat destined for human consumption. The tested amounts were, respectively, 50 and 500 mg of melamine/kg of feed. The addition of melamine had no significant effect on feed consumption and egg production. However, melamine appeared in the eggs as early as the first day of exposure. The average concentration was reached after the third day at both levels of contamination. The amounts of melamine found in eggs and tissues were almost directly proportional to the quantities ingested. However, melamine did not appear to accumulate in the organs and tissues that were studied.

First full-length sequences of the S gene of European isolates reveal further diversity among turkey coronaviruses.

An increasing incidence of enteric disorders clinically suggestive of the poult enteritis complex has been observed in turkeys in France since 2003. Using a newly designed real-time reverse transcriptase-polymerase chain reaction assay specific for the nucleocapsid (N) gene of infectious bronchitis virus (IBV) and turkey coronaviruses (TCoV), coronaviruses were identified in 37% of the intestinal samples collected from diseased turkey flocks. The full-length spike (S) gene of these viruses was amplified, cloned and sequenced from three samples. The French S sequences shared 98% identity at both the nucleotide and amino acid levels, whereas they were at most 65% and 60% identical with North American (NA) TCoV and at most 50% and 37% identical with IBV at the nucleotide and amino acid levels, respectively. Higher divergence with NA TCoV was observed in the S1-encoding domain. Phylogenetic analysis based on the S gene revealed that the newly detected viruses form a sublineage genetically related with, but significantly different from, NA TCoV. Additionally, the RNA-dependent RNA polymerase gene and the N gene, located on the 5' and 3' sides of the S gene in the coronavirus genome, were partially sequenced. Phylogenetic analysis revealed that both the NA TCoV and French TCoV (Fr TCoV) lineages included some IBV relatives, which were however different in the two lineages. This suggested that different recombination events could have played a role in the evolution of the NA and Fr TCoV. The present results provide the first S sequence for a European TCoV. They reveal extensive genetic variation in TCoV and suggest different evolutionary pathways in North America and Europe.

Development and validation of four Real-Time quantitative RT-PCRs specific for the positive or negative strands of a bisegmented dsRNA viral genome.

Four tagged quantitative Real-Time RT-PCRs (qRT-PCRs) were developed to quantify the positive and negative strands of segments A and B of the bisegmented double-stranded RNA (dsRNA) genome of infectious bursal disease virus (IBDV, family Birnaviridae, genus Avibirnavirus). The qRT-PCRs were validated using single-stranded RNAs corresponding to each genomic strand (A+, B+, A-, B-). Specific quantitation proved possible from 5×10(7) to 5×10(2) copies of the template per reaction, with excellent reproducibility and linearity. The methods detected similar amounts of A+ and A- and of B+ and B- in a purified dsRNA viral genome preparation, thus corroborating the accuracy of quantitation. The qRT-PCRs were used to quantify the four strands in CsCl purified virus fractions and in samples collected during propagation of IBDV in cell culture. Purified virus fractions contained similar amounts of A- and B- strands, but also a large and unexplained excess of A+ and even more B+ strands. Results of the in vitro kinetic study showed an early accumulation of positive strands and a more delayed and lower accumulation of the A- and B- strands, both in similar amounts. These results suggest that minus strand synthesis occurs in IBDV after the equimolar packaging of A+ and B+ strands.

Laboratory evaluation of a quantitative real-time reverse transcription PCR assay for the detection and identification of the four subgroups of avian metapneumovirus.

Avian metapneumovirus (AMPV) is an important pathogen causing respiratory diseases and egg drops in several avian species. Four AMPV subgroups have been identified. The laboratory diagnosis of AMPV infections relies on serological methods, on labour-intensive virus isolation procedures, and on recently developed subgroup specific reverse transcription PCR (RT-PCR) protocols. In the present study, both the specificity and sensitivity of a commercial real-time reverse transcription PCR (RRT-PCR) for the detection and identification of the four AMPV subgroups were evaluated. Fifteen non-AMPV avian viruses belonging to 7 genera and 32 AMPV belonging to the 4 subgroups were tested. No non-AMPV virus was detected, whereas all AMPV viruses were identified in agreement with their previous molecular and antigenic subgroup assignment. The sensitivity and quantitating ability of the RRT-PCR assay were determined using serial dilutions of RNA derived either from AMPV virus stocks or from runoff transcripts. In all cases, linear dose/responses were observed. The detection limits of the different subgroups ranged from 500 to 5000 RNA copies and from 0.03 to 3.16TCID50/ml. The results were reproducible under laboratory conditions, thus showing that quantitative RRT-PCR is a new and powerful tool for the rapid and sensitive detection, identification and quantitation of AMPVs.

Chicken recombinant antibodies specific for very virulent infectious bursal disease virus.

A phage-displayed single chain variable fragment (scFv) antibody library was constructed from the immune spleen cells of chickens immunized with very virulent infectious bursal disease virus (vvIBDV) strain CS89. A library consisting of around 9.2 x 10(7) clones was subjected to 3 rounds of panning against captured CS89 virus. Analysis of individual clones by nucleotide sequencing revealed at least 22 unique scFv antibodies binding to vvIBDV in ELISA. Testing of the scFv antibody panel in ELISA against classical, variant or vaccine strains and a wide variety of vvIBDV isolates from the UK, China, France, Belgium, Africa, Brazil, Indonesia and the Netherlands identified one antibody, termed chicken recombinant antibody 88 (CRAb 88) that was specific for vvIBDV. CRAb 88 was capable of recognizing all vvIBDV strains tested regardless of their country of origin and showed no reactivity with classical, variant or vaccine strains, lending support to the use of this scFv as a powerful diagnostic tool for the differentiation of vvIBDV strains. Immunoprecipitation studies revealed that CRAb 88 was directed towards a highly conformational epitope located within the major neutralizing protein VP2. Sequence analysis of the hypervariable region of VP2 of the IBDV strains tested indicate that Ile(256) and Ile(294) may play roles in binding of CRAb 88. This is the first reagent of its type capable of positively distinguishing vvIBDV from other IBDV strains.

European and American subgroup C isolates of avian metapneumovirus belong to different genetic lineages.

The gene encoding the attachment glycoprotein (G) was sequenced in three French isolates of-subgroup C avian metapneumovirus (APV-C) from ducks. With 1771 nt, this gene proved as long as recently published for North-American APV-C isolates from turkeys. The nt sequences of the duck viruses shared 99% identity but proved only 75-83% identical with their North-American counterparts, viruses of both origins encoding 585 amino acid (aa)-long G proteins. Alignments revealed more homogeneity within the European and North-American groups (at least 98 and 79% aa identity, respectively) than between European and North-American viruses (at best 70% a identity), and confirmed the presence of an extracellular divergent domain (positions 302-484) in APV-C G. A phylogenetic analysis demonstrated that North-American and French isolates of APV-C belonged to significantly different genetic lineages, in agreement with the different geographical origin and host species of these viruses.

Significance of the genetic relationships deduced from partial nucleotide sequencing of infectious bursal disease virus genome segments A or B.

The rapid genomic characterization of infectious bursal disease virus (IBDV) requires determining which partial nucleotide (nt) sequences derived from IBDV segments A or B would produce phylogenetic information as significant as sequencing the whole corresponding segments. Long nt coding sequences of 27 IBDV segments A (aa 20-991) and 21 segments B (aa 7-stop codon) were retrieved from databanks and used to compute reference phylogenetic trees using Neighbor Joining (NJ) and Parsimony (P): clusters appearing in the NJ and P reference trees with a bootstrap value greater than 80% were considered as significant (Whole Segment Clusters, WSC). The sequences were then cut into overlapping regions. These were used to compute phylogenetic trees which were compared with reference ones. Of the partial sequences, the VP2 gene best represented IBDV segment A (10 out of 13 WSC were conserved), and the 5' two thirds of segment B best represented segment B (5 to 6 conserved WSC out of 6). Implementation of the Plato programme finally demonstrated that the region encoding VP2 variable domain (vVP2, segment A) is the only region of IBDV genome with a significantly different evolution rate, which result is consistent with vVP2 being subjected to a high selection pressure.