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The SARS-CoV-2 spike protein Q677P/H mutation and furin cleavage site (FCS) have been shown to affect cell tropism and virus transmissibility. Here, we analyzed the frequency of Q677P/H and FCS point mutations in 1,144,793 human and 1042 animal spike protein sequences and from those of the emergent variants B.1.1.7, B.1.351, P.1, B.1.429 + B.1.427, and B.1.525, which were deposited in the database of the GISAID Initiative. Different genetic polymorphisms, particularly P681H and A688V, were detected in the FCS, mainly in human isolates, and otherwise, only pangolin and bat sequences had these mutations. Multiple FCS amino acid deletions such as Δ680SPRRA684 and Δ685RSVA688 were only detected in eight and four human isolates, respectively. Surprisingly, deletion of the entire FCS motif as Δ680SPRRARSVA688 and Δ680SPRRARSVAS689 was detected only in three human isolates. On the other hand, analysis of FCS from emergent variants showed no deletions in the FCS except for spike P681del, which was detected in seven B.1.1.7 isolates from the USA. Spike Q677P was detected only once in variant, B.1.1.7, whereas Q677H was detected in all variants, i.e., B.1.1.7 (n = 1938), B.1.351 (n = 28), P.1 (n = 9), B.1.429 + B.1.427 (n = 132), and B.1.525 (n = 1584). Structural modeling predicted that mutations or deletions at or near the FCS significantly alter the cleavage loop structure and would presumably affect furin binding. Taken together, our results show that Q677H and FCS point mutations are prevalent and may have various biological effects on the circulating variants. Therefore, we recommend urgent monitoring and surveillance of the investigated mutations, as well as laboratory assessment of their pathogenicity and transmissibility.

Although chickens are not susceptible to SARS-CoV-2, several coronavirus disease outbreaks have been described concerning poultry processing facilities in different countries. The COVID-19 pandemic and the developed strain caused 2nd, 3rd, and recent Indian strain waves of epidemics that have led to unexpected consequences, such as forced reductions in demands for some industries, transportation systems, employment, and businesses due to public confinement. Besides, poultry processing plants' conditions exacerbate the risks due to the proximity on the line, cold, and humidity. Most workers do not have access to paid sick time or adequate health care, and because of the low wages, they have limited reserves to enable them to leave steady employment. In addition, workers in meat and poultry slaughterhouses may be infected through respiratory droplets in the air and/or from touching dirty surfaces or objects such as workstations, break room tables, or tools. Egg prices have increased dramatically during the lockdown as consumers have started to change their behaviors and habits. The COVID pandemic might also substantially impact the international poultry trade over the next several months. This review will focus on the effect of COVID-19 on poultry production, environmental sustainability, and earth systems from different process points of view.

Avian influenza virus (AIV) variants emerge frequently, which challenges rapid diagnosis. Appropriate diagnosis reaching the sub- and pathotype level is the basis of combatting notifiable AIV infections. Real-time RT-PCR (RT-qPCR) has become a standard diagnostic tool. Here, a total of 24 arrayed RT-qPCRs is introduced for full subtyping of 16 hemagglutinin and nine neuraminidase subtypes of AIV. This array, designated Riems Influenza A Typing Array version 2 (RITA-2), represents an updated and economized version of the RITA-1 array previously published by Hoffmann et al. RITA-2 provides improved integration of assays (24 instead of 32 parallel reactions) and reduced assay volume (12.5 µL). The technique also adds RT-qPCRs to detect Newcastle Disease (NDV) and Infectious Bronchitis viruses (IBV). In addition, it maximizes inclusivity (all sequences within one subtype) and exclusivity (no intersubtypic cross-reactions) as shown in validation runs using a panel of 428 AIV reference isolates, 15 reference samples each of NDV and IBV, and 122 clinical samples. The open format of RITA-2 is particularly tailored to subtyping influenza A virus of avian hosts and Eurasian geographic origin. Decoupling and re-arranging selected RT-qPCRs to detect specific AIV variants causing epizootic outbreaks with a temporal and/or geographic restriction is possible.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is currently spreading worldwide. The pandemic has already had significant adverse effects on human civilization, the environment, and the ecosystem at national and global levels. Moreover, the various sectors of the food production chain, particularly agriculture and livestock, have also been significantly affected in terms of production sustainability and economic losses. The global pandemic has already resulted in a sharp drop in meat, milk, and egg production. Restrictions of movement at national and international levels, implemented as a part of control strategies by public health sectors, have negatively impacted business related to the supply of raw materials for livestock farmers and farm outputs, veterinary services, farmworkers, and animal welfare. This review highlights the significant impacts of COVID-19 on the sustainability of livestock performance, welfare on a global scale, and strategies for mitigating these adverse effects.

Background Newcastle disease is a devastating disease in poultry caused by virulent Newcastle disease virus (NDV), a paramyxovirus endemic in many regions of the world despite intensive vaccination. Phylogenetic analyses reveal ongoing evolution of the predominant circulating genotype 2.VII, and the relevance of potential antigenic drift is under discussion. To investigate variation within neutralization-sensitive epitopes within the protein responsible for receptor binding, i.e. the Hemagglutinin-Neuraminidase (HN) spike protein, we were interested in establishing genotype-specific monoclonal antibodies (MAbs). Methods An HN-enriched fraction of a gradient-purified NDV genotype 2.VII was prepared and successfully employed to induce antibodies in BalbC mice that recognize conformationally intact sites reactive by haemagglutination inhibition (HI). For subsequent screening of mouse hybridoma cultures, an NDV-ELISA was established that utilizes Concanavalin A (ConA-ELISA) coupled glycoproteins proven to present conformation-dependent epitopes. Results Six out of nine selected MAbs were able to block receptor binding as demonstrated by HI activity. One MAb recognized an epitope only present in the homologue virus, while four other MAbs showed weak reactivity to selected other genotypes. On the other hand, one broadly cross-reacting MAb reacted with all genotypes tested and resembled the reactivity profile of genotype-specific polyclonal antibody preparations that point to minor antigenic differences between tested NDV genotpyes. Conclusions These results point to the concurrent presence of variable and conserved epitopes within the HN molecule of NDV. The described protocol should help to generate MAbs against a variety of NDV strains and to enable in depth analysis of the antigenic profiles of different genotypes.

Low pathogenic avian influenza H9N2 is still circulating in the Middle East causing respiratory manifestations and severe economic losses in poultry. In the present study, an H9 plasmid-based DNA vaccine targeting the HA gene of H9N2 A/CK/Egypt/SCU8/2014 was developed and evaluated in turkeys. The full length of HA was cloned into vector plasmids under the control of a cytomegalovirus promoter. The in-vitro expression of the recombinant HA was demonstrated in HeLa cells transfected with the plasmids pVAX1-H9 or pCR-H9 using western blot and Immunofluorescent assay (IFA). The efficacy of pVAX-H9 and pCR- H9, naked or saponin-adjuvanted, was evaluated in turkey poults at 3 weeks and challenged with A/CK/Egypt/SCU8/2014 (106 EID50/bird at 3 weeks post-vaccination. The efficacy was assesses based on virus shedding, oropharyngeal and cloacal, as well as seroconversion using haemagglutination inhibition (HI) test. All immunized birds showed high HI antibody titers (7-8 log2) at 3 weeks post-vaccination. None of the birds vaccinated with naked or saponin-adjuvanted pVAX-H9 or pCR-H9 showed any clinical signs. The pVAX-H9 and pCR-H9 alone did not prevent cloacal and oropharyngeal virus shedding, however, saponin-adjuvanted pVAX1-H9 and pCR-H9 prevented cloacal and oropharyngeal virus shedding at 3 and 5 days post challenge, respectively. In conclusion, DNA vaccination with pVAX1-H9 and pCR-H9 could protect turkey from the H9N2 virus, but vaccination regimes need to be improved.

The avian pathogen Ornithobacterium rhinotracheale (ORT) has been implied in the etiology of poultry respiratory disease in recent years. To evaluate whether Whatman® Flinders Technology Associates (FTA®) cards can be used for hazard-free transport and storage of ORT samples for posterior DNA amplification, a controlled assay was performed. Three 10-fold dilutions of an ORT culture suspension were spotted on FTA cards and stored at room temperature (RT) for 6 mo. Sterile swabs were immersed in the same three 10-fold culture dilutions and stored at RT and 4 and -20 C without storage medium for the same time. DNA was extracted from both the FTA cards and swabs 1 day, 1 and 6 wk, and 6 mo following sample preparation and stored at -20 C. At the end of the experiment, real-time PCR amplification of the 16S ribosomal RNA gene was performed from DNA extracted throughout a 6-mo period from all ORT samples stored on both FTA cards and swabs. The obtained threshold cycle values for each ORT DNA extraction date were within the same range for all samples in a dilution-dependent fashion, regardless of storage temperature or used material. Pure ORT colonies could be reisolated 1 day after sample preparation from the swab dilutions stored at all temperatures but not from the FTA cards. We conclude that the efficiency of ORT DNA amplification from samples stored on FTA cards or in swabs is similar. However, FTA cards have the advantage of preventing microorganism growth, thus allowing safe transport and storage, for at least 6 mo, for bacterial dilutions down to at least 104-105 colony-forming units/ml.

Background H9N2 avian influenza virus is endemic in Egyptian poultry flocks. The role of the live viral vaccines such as LaSota in exaggeration of the clinical picture of H9N2 infection under field conditions is significantly important leading to severe economic losses due to higher mortality and lower growth performance. This experiment was designed to identify the possible interaction between experimental infection with H9N2 virus and NDV live vaccine (LaSota strain) in broiler chickens. Six groups each of 20 broiler chicks were used. Three groups (G1–3) were infected with H9N2 and vaccinated with LaSota, 3 days before, at the same day or 3 days post vaccination (dpv), while the remaining groups (G4–6) were non-vaccinated infected, vaccinated non-infected and non-vaccinated non-infected. Results The highest mortality rate (37.5%) was noticed in chickens of G1 (H9N2 infected 3 days prior LaSota vaccination). Also, this bird group had the most severe clinical signs, histopathological lesions and the longest viral shedding for 9 days post infection (dpi). In the 2nd and 3rd groups, the mortality rate was the similar (31.2%) with less pronounced clinical signs, histopathological lesions and H9N2 shedding was for only 6 dpi with the least shedding quantity in chickens of G3. The control non-vaccinated infected chickens (G4) had 18.7% mortality with the least degree of clinical signs, lesions and the highest viral shedding quantity but only for 6 dpi. At 35 days of age, there was a statistical significant decrease (P

Highlights • Development of molecular diagnostic tools for infectious bronchitis virus (IBV). • Segregation of the IBV Egyptian variant II into two phylogroups detected. • Frequent co-infections with IBV, avian influenza and Newcastle disease observed., In Egypt, currently two geographically restricted genotypes of the infectious bronchitis coronavirus (IBV) are circulating with detrimental effects for poultry industry. A sensitive real-time RT-PCR assay targeting the IBV nucleocapsid gene (N) was developed to screen clinical samples for presence of IBV. Conventional RT-PCRs amplifying hypervariable regions (HVRs 1–2 and 3) of the IBV S1 gene were developed and amplificates used for nucleotide sequence-based typing of IBV field strains in Egyptian chickens directly from clinical samples.

The Mycoplasma strain ARNO was isolated from the semen of a clinically healthy gyrfalcon (Falco rusticolus). Colonies of strain ARNO grew in fried-egg shape on Mycoplasma agar plates (SP4). The organism did not ferment glucose or hydrolyze arginine or urea; hence, organic acids are assumed as energy source. Growth was sterol-dependent and optimal growth temperature 42 °C, with a temperature range from 20 to 44 °C. Strain ARNO was not identified as a representative of any of the currently described Mycoplasma species by alignment of the 16S rRNA gene sequence and 16 S–23 S intergenic transcribed spacer region, or immunobinding assay. Hence, strain ARNO represents a novel Mycoplasma species for which the name Mycoplasma seminis sp. nov. is proposed (DSM 27653, NCTC 13927). After developing a species-specific PCR, the prevalence of M. seminis sp. nov. was determined in adult and juvenile falcons in a commercial breeding center for falcons. Semen samples (n = 171) were obtained from 113 male adults, due to repeated sampling of 39 birds. Female adults (n = 26) were sampled once, while 105 of the 152 juvenile birds were sampled twice via choanal swabs. Mycoplasma seminis sp. nov. was found in the semen of clinically healthy adult males (3.5 %) as well as in the respiratory tract of female (34.6 %) and juvenile birds (59.2 %). After comparison of semen samples with (2.9 %) and without M. seminis sp. nov. identification, no indications for a potential influence on the semen quality were demonstrated. Hence, M. seminis sp. nov. seems likely to be of commensal character in falcons.

Highlights • New prototype of emerging IBV variant recognized in Egypt. • New IBV strains form a forth subclade within S-genotype GI-23. • Mutations in the S1protein, including two defined neutralizing sites, point to antigenic variation. • Intra-genotypic recombination is recognized for genes S1, 3ab and E, inter-genotypic recombination for gene 6b (GI-19) • Specific gene 3ab genotype is linked to new IBV emergence of new variants., Infectious bronchitis virus (IBV), a gamma-coronavirus, causes infectious bronchitis (IB), a major respiratory disease of chicken. Its high mutation rate in conjunction with recombination of the RNA genome constantly creates IBV variants that are difficult to control by currently available vaccines. In this study, we addressed the question whether small-scale holdings might harbor IBV variants that serve as a reservoir for newly emerging variants. Egyptian IBV isolate EGY/NR725/2016 (NR725/16) from a small-scale broiler farm was assigned to genotype I, clade 23 (S1:GI-23), based on partial S1 gene sequences and corroborated by full genome sequencing. Analysis of the S1 gene established three subclades for historical IBV strains (S1:GI-23.1, S1:GI-23.2.1 and S1:GI-23.2.2) and confirmed NR725/16 as being part of a separate fourth subclade (S1:GI-23.3). Samples from the years 2018 and 2019 revealed that the new subclade prevails in Egypt, carrying fixed mutations within the hypervariable regions (HVR) 1-3 of the S1 protein that affect two neutralization sensitive epitopes at sites 294F, 297S and 306Y (48.2) and 329R (62.1). In addition, recombination was recognized in isolate NR 725/16, with intra-subtype mixing for the entire genes 3ab and E and inter-subtype mixing for the entire gene 6b with a close match to QX like viruses of genotype GI-19. Further analysis of gene 3ab detected the homologous gene pool to NR725/16 in samples from 2013 (3ab:C) and closely related 3ab genotypes in IBV Egyptian isolates from 2016, 2018 and 2019. These data prove a flourishing exchange between poultry holdings with a common gene pool. The continued circulation of viruses harboring genes S1:GI-23.3 and 3ab:C indicates an evolutionary advantage of this combination possibly by combining antigenic escape with modulated pathogenicity to facilitate IBV spread in the vaccinated poultry population in Egypt.

Highly pathogenic (HP) H5N1, clade 2.2.1, and low pathogenic avian influenza (LPAI) H9N2 viruses, G1-B lineage, are endemic in poultry in Egypt and have co-circulated for almost a decade. Surprisingly, no inter-subtypic reassortment events have been reported from the field during that time. After the introduction of HPAIV H5N8, clade 2.3.4.4b, in Egyptian poultry in 2016, suddenly HP H5N2 reassortants with H9N2 viruses emerged. The current analyses focussed on studying 32 duck flocks, 4 broiler chicken flocks, and 1 turkey flock, suffering from respiratory manifestations with moderate to high mortality reared in two Egyptian governorates during 2019. Real-time RT-PCR substantiated the presence of HP H5N8 in 21 of the 37 investigated flocks with mixed infection of H9N2 in two of them. HP H5N1 was not detected. Full hemagglutinin (HA) sequencing of 10 samples with full-genome sequencing of three of them revealed presence of a single genotype. Very few substituting mutations in the HA protein were detected versus previous Egyptian HA sequences of that clade. Interestingly, amino acid substitutions in the Matrix (M2) and the Neuraminidase (NA) proteins associated with conferring both Amantadine and Oseltamivir resistance were present. Systematic reassortment analysis of all publicly available Egyptian whole genome sequences of HP H5N8 (n = 23), reassortant HP H5N2 (n = 2) and LP H9N2 (n = 53) viruses revealed presence of at least seven different genotypes of HPAI H5Nx viruses of clade 2.3.4.4b in Egypt since 2016. For H9N2 viruses, at least three genotypes were distinguishable. Heat mapping and tanglegram analyses suggested that several internal gene segments in both HP H5Nx and H9N2 viruses originated from avian influenza viruses circulating in wild bird species in Egypt. Based on the limited set of whole genome sequences available, annual replacement patterns of HP H5Nx genotypes emerged and suggested selective advantages of certain genotypes since 2016.

Ornithobacterium rhinotracheale (ORT) is an important bacterial pathogen of great economic significance to poultry production. This bacterium causes severe disease in chickens and turkeys worldwide. The objective of this study was to characterize ORT isolates from two different geographic locations in the United States by multilocus sequence typing (MLST). A total of 60 isolates were included in this study; 36 from California and 24 from Minnesota. All 60 isolates were confirmed to be ORT by PCR that targeted the 16S rRNA gene. The results of MLST revealed eight different sequence types (ST) of ORT. Out of these, four were novel and were assigned numbers ST-32, ST-33, ST-34, and ST-35. ST-1 was the predominant sequence type among all isolates followed by ST-9 and ST-8. Only one isolate was identified as ST-2. No significant variation was seen in STs in ORT isolated from different years. In turkeys, 76.3% (29/38) of isolates belonged to ST-1 and 7.9% (3/38) to ST-8. Of the chicken isolates, 72.2% (13/18) belonged to ST-1 and 16.6% (3/18) to ST-9. Isolates from both states showed low genetic variability. Of the 32 isolates from California, 24 (75%) were identified as ST-1 and 4 (12.5%) were identified as ST-9. The most prevalent sequence type was ST-1 (17/24) followed by ST-8 (3/24) in Minnesota. Three isolates from turkeys in Minnesota belonged to the same ST (ST-8) as the already known ORT strain RefO, which isolated from a rook in Germany in 2000. Whether this sequence type had evolved from wild birds could not be ascertained in this study.

Continuous loading of the skeleton by the body’s weight is an important factor in establishing and maintaining bone morphology, architecture and strength. However, in fast-growing chickens the appendicular skeleton growth is suboptimal making these chickens predisposed to skeletal mineralization disorders and fractures. This study compared the macro- and microstructure as well as the mechanical properties of the tibiotarsus of a novel dual-purpose, Lohmann Dual (LD) and a highly developed broiler, Ross (Ross 308) chicken line. Eighty one-day-old male chicks of each line were grown until their body weight (BW) reached 2000g. Starting at the day of hatching, six birds of each line were sampled weekly. The weight, length and width of the tibiotarsus were measured and its mechanical properties (rigidity, M-Max and the M-fracture) were evaluated using the three-point bending test. Additionally, the mineral density of both, trabecular and cortical bone, the bone volume fraction, the trabecular number, thickness and separation plus cortical thickness of both chicken lines were analyzed using microcomputed tomography. The growth of the tibiotarsus in both chicken lines followed a similar pattern. At the same age, the lighter LD chickens had shorter, thinner and lighter tibiotarsi than those of Ross chickens. However, the LD chickens had a similar cortical thickness, bone volume fraction and similar mineral density of both trabecular and cortical bone to that of Ross chickens. Furthermore, the tibiotarsus of LD chickens was longer, heavier and wider than those of Ross chickens of the same BW. In addition the rigidity of the LD tibiotarsus was greater than that of Ross chickens. This suggests that the tibiotarsus of LD chickens had more bending resistance than those of Ross chickens of the same BW. Consequently, fattening LD chickens to the marketable weight should not affect their leg skeleton stability.

The presence of extended-spectrum beta-lactamase (ESBL) producing Escherichia coli on poultry products is an important issue for veterinary and human health due to the zoonotic infection risk for producers and consumers. The present study focuses on testing the efficacy of six different disinfection methods on eggshell samples, aiming to reduce ESBL producing E. coli contamination on the hatching egg. Sterile eggshell cutouts were artificially contaminated with 10(8) cfu/ml CTX-M-1 producing E. coli and used as a carrier model to analyze the efficacy of six disinfection methods. The contaminated samples were separated into two groups; 1) contaminated and disinfected, 2) contaminated and non-disinfected. Six independent disinfection protocols were performed following product specifications and protocols. Each eggshell sample was separately crushed, and the total viable bacterial count was calculated to determine the disinfection efficacy. Five out of six tested methods (formaldehyde gassing, hydrogen peroxide + alcohol spray, essential oils spray, peracetic acid foam, and low energetic electron radiation) demonstrated a reduction or completely eliminated the initial ESBL producing E. coli contamination. One method (essential oils as cold fog) only partly reached the expected efficacy threshold (reduction of >10(2) cfu/ml) and the result differed significantly when compared to the reference method i.e. formaldehyde gassing.

Virulence-associated traits have frequently been studied in enterococci and are considered to contribute towards the pathogenicity of infections. In the present study, Enterococcus isolates were collected during diagnostic investigations from meat turkeys in Germany. Twenty-eight isolates of three different Enterococcus species were analyzed for five selected putative virulence traits to understand their potential role in the pathogenicity using the chicken embryo lethality assay. Ten E. faecalis, ten E. faecium, and eight E. gallinarum isolates were examined for the presence of common virulence genes and their phenotypic expression, namely, the cytolysin operon, five individual cyl genes (cylLL, cylLS, cylM, cylB, and cylA), gelatinase (gelE), hyaluronidase (hylEfm), aggregation substance (asa1), and enterococcal surface protein (esp). The Enterococcus isolates showed significant species-dependent differences in the presence of genotypic traits (p < 0.001 by Fisher's exact test; Cramer's V = 0.68). At least one gene and up to three virulence traits were found in E. faecalis, while six E. faecium isolates and one E. gallinarum isolate did not display any virulence-associated pheno- or genotype. More than half of the Enterococcus isolates (n = 15) harbored the gelE gene, but only E. faecalis (n = 10) expressed the gelatinase activity in vitro. The hylEfm gene was found in five E. gallinarum isolates only, while seven isolates showed the hyaluronidase activity in the phenotypic assay. In Cramer's V statistic, a moderate association was indicated for species (V ≤ 0.35) or genotype (V

During routine diagnosis in 2012, 69 samples of diseased turkey breeding and fattening flocks in Germany were examined for infection with aviadenoviruses by virus isolation using primary chicken embryo liver cells. In total, 21 aviadenovirus isolates, identified by a group-specific indirect immunofluorescence test, were obtained from 19 flocks. In almost all cases, molecular typing of these isolates based on partial hexon gene sequences revealed the presence of different types of turkey aviadenoviruses (TAdVs), including species Turkey aviadenovirus B (TAdV-B) with at least two different genotypes, as well as the species Turkey aviadenovirus C (TAdV-C) and Turkey aviadenovirus D (TAdV-D). Further analysis of DNA-dependent DNA polymerase gene sequences confirmed the classification of selected TAdV-C and TAdV-D isolates. Based on the results obtained for both genes, we suggest that TAdV-2, in addition to TAdV-4, belongs to the species TAdV-C. In contrast, amplification of the DNA polymerase gene fragment of nearly all investigated TAdV-B isolates failed due to unknown reasons. The results of sequence and phylogenetic analysis support the previously proposed classification of TAdVs into three different species and demonstrated how widely spread these viruses are in German turkey flocks. Analysis of case histories revealed a wide range of clinical and pathological changes; however an apparent link between types and disease conditions was not identified.

Virulence-associated traits have frequently been studied in enterococci and are considered to contribute towards the pathogenicity of infections. In the present study,Enterococcusisolates were collected during diagnostic investigations from meat turkeys in Germany. Twenty-eight isolates of three differentEnterococcusspecies were analyzed for five selected putative virulence traits to understand their potential role in the pathogenicity using the chicken embryo lethality assay. TenE. faecalis, tenE. faecium, and eightE. gallinarumisolates were examined for the presence of common virulence genes and their phenotypic expression, namely, the cytolysin operon, five individualcylgenes (cylLL,cylLS,cylM,cylB, andcylA), gelatinase (gelE), hyaluronidase (hylEfm), aggregation substance (asa1), and enterococcal surface protein (esp). TheEnterococcusisolates showed significant species-dependent differences in the presence of genotypic traits (p<0.001by Fisher’s exact test; Cramer’sV = 0.68). At least one gene and up to three virulence traits were found inE. faecalis, while sixE. faeciumisolates and oneE. gallinarumisolate did not display any virulence-associated pheno- or genotype. More than half of theEnterococcusisolates (n = 15) harbored thegelEgene, but onlyE. faecalis(n = 10) expressed the gelatinase activityin vitro. ThehylEfmgene was found in fiveE. gallinarumisolates only, while seven isolates showed the hyaluronidase activity in the phenotypic assay. In Cramer’sVstatistic, a moderate association was indicated for species (V ≤ 0.35) or genotype (V E. gallinarumisolates were less virulent with mortality rates ranging between 0 and 30%. TwoE. faecalisisolates were highly virulent, harboring the wholecyl-operon as well asgelEandasa1genes. Likewise, oneE. faeciumisolate caused high embryo mortality but did not harbor any of the investigated virulence genes. For the first time,Enterococcusisolates of three different species collected from diseased turkeys were investigated for their virulence properties in comparison. The results differed markedly between theEnterococcusspecies, withE. faecalisharboring the majority of investigated genes and virulence traits. However, the genotype did not entirely correlate with the phenotype or the isolates’ virulence potential and pathogenicity for chicken embryos.

Rearing dual-purpose chickens is a practicable approach to avoid culling one-day-old male layer chicks. The present study examined the impact of a conventional fattening diet on the liver of a novel dual-purpose chicken line (Lohmann Dual, LD) in comparison to a broiler (Ross 308) chicken line. Age-related changes of structure and lipid content of the liver were assessed. One hundred twenty and newly hatched chicks (LD = 66, Ross = 54) were kept under the same husbandry conditions and fed a commercial diet for 5 weeks for Ross and 9 weeks for LD. Six birds of each line were examined weekly. Their body weight (BW) and liver mass were recorded. Microscopic structure and ultrastructure of the liver were investigated and the liver lipid content was measured using a pre-validated method. During the study period, liver mass increased with age, while normalized liver mass decreased. Furthermore, liver mass of Ross birds was greater than that of LD birds of the same BW. Overall, no significant differences were observed in the hepatic structure or ultrastructure between the two chicken lines. The hepatic lymphatic aggregations were without fibrous capsules and their number and area increased throughout the first week, then the values began to fluctuate with age in both chicken lines. The changes in the liver lipid content of the two chicken lines were within the normal physiological range over the term of the study. The liver lipid content correlated negatively with age and body weight in both lines. It was the highest on the first day then decreased until day 7 and thereafter did not change in both chicken lines. However, given the same body weight, the Ross chickens had a 9% greater liver lipid content than LD chickens. It is concluded that there is no apparent adverse effect of a high-energy diet on the liver of LD chickens.

Background Vaccination of poultry to control highly pathogenic avian influenza virus (HPAIV) H5N1 is used in several countries. HPAIV H5N1 of clade 2.2.1 which is endemic in Egypt has diversified into two genetic clades. Clade 2.2.1.1 represents antigenic drift variants in vaccinated commercial poultry while clade 2.2.1.2 variants are detected in humans and backyard poultry. Little is known about H5N1 infection in vaccinated turkeys under field conditions. Case presentation Here, we describe an HPAI H5N1 outbreak in a vaccinated meat-turkey flock in Egypt. Birds were vaccinated with inactivated H5N2 and H5N1 vaccines at 8 and 34 days of age, respectively. At 72nd day of age (38 days post last vaccination), turkeys exhibited mild respiratory signs, cyanosis of snood and severe congestion of the internal organs. Survivors had a reduction in feed consumption and body gain. A mortality of ~29% cumulated within 10 days after the onset of clinical signs. Laboratory diagnosis using RT-qPCRs revealed presence of H5N1 but was negative for H7 and H9 subtypes. A substantial antigenic drift against different serum samples from clade 2.2.1.1 and clade 2.3.4.4 was observed. Based on full genome sequence analysis the virus belonged to clade 2.2.1.2 but clustered with recent H5N1 viruses from 2015 in poultry in Israel, Gaza and Egypt in a novel subclade designated here 2.2.1.2a which is distinct from 2014/2015 2.2.1.2 viruses. These viruses possess 2.2.1.2 clade-specific genetic signatures and also mutations in the HA similar to those in clade 2.2.1.1 that enabled evasion from humoral immune response. Taken together, this manuscript describes a recent HPAI H5N1 outbreak in vaccinated meat-turkeys in Egypt after infection with a virus representing novel distinct 2.2.1.2a subclade. Conclusions Infection with HPAIV H5N1 in commercial turkeys resulted in significant morbidity and mortality despite of vaccination using H5 vaccines. The isolated virus showed antigenic drift and clustered in a novel cluster designated here 2.2.1.2a related to viruses in poultry in Israel, Gaza and Egypt. Enforcement of biosecurity and constant update of vaccine virus strains may be helpful to protect vaccinated birds and prevent spillover infection to neighbouring countries. Electronic supplementary material The online version of this article (doi:10.1186/s12985-017-0697-5) contains supplementary material, which is available to authorized users.

Avian bornavirus (ABV) has been identified as the cause of proventricular dilatation disease in birds, but the virus is also found in healthy birds. Most studies of ABV have focused on captive birds. We investigated 86 free- ranging psittacine birds in Brazil and found evidence for natural, long-term ABV infection.

Several studies have shown differences in the course of histomonosis, the infection with the trichomonad parasite Histomonas meleagridis, in different chicken breeds. In the present study, 10 specific-pathogen-free (SPF) layer-type (LT) chickens and twelve SPF meat-type (MT) chickens were infected intracloacally with 200,000 H. meleagridis trophozoites. One and two weeks postinfection (p.i.), three birds of each group were euthanatized. The remaining birds were euthanatized 3 wk p.i. Infected birds showed severe gross lesions typical for histomonosis in ceca at the first and second week p.i., while livers showed necrotic foci at 2 and 3 wk p.i., but only very rarely at 1 wk p.i. Differences between groups in the severity of lesions were statistically insignificant. In histopathology, LT chickens showed a significantly more-severe necrosis and ablation of the cecal epithelium 1 wk p.i. Parasites without inflammation were also found in most investigated spleens and lungs but only in a few kidneys. Investigation of these organs for histomonal DNA by real-time PCR confirmed these results. In addition, the humoral immune response against histomonal actinin 1 and 3 was measured by an ELISA. The humoral immune response against actinin 1 started sooner and was significantly higher in LT chickens than in MT chickens. In conclusion, the results of the present study suggest that the genetic background of the birds influences the reaction to infection with H. meleagridis.

Between 2006 and 2011 a series of disease conditions characterized by raised mortality and liver disorders occurred in turkey breeder flocks and in meat turkey flocks in Germany. The flocks were between 12 and 23 wk of age, and mostly hens were affected. Clinical signs were nonspecific and accompanied by mortality varying between 1% and 7%. Affected birds displayed swollen livers that were marbled with black and red spots and yellowish areas. The pericardium was filled with an amber fluid, and the coronary groove was extensively filled with fat. Spleens were swollen, and a serous fluid that seemed to leak from the liver was present in the body cavity. Histopathological findings in all but one case included fatty degeneration of hepatocytes with parenchymal collapse and associated hemorrhages. Some animals showed cholangitis and hepatitis with intranuclear inclusion bodies. In three cases with breeders, electron microscopy detected virus particles that were between 23 and 30 nm and similar to parvo- or picornavirus. In addition, picornavirus RNA was detected in the livers of one meat turkey flock. Investigations by PCR for circovirus, polyomavirus parvovirus, and aviadenovirus yielded negative results in all cases, but an aviadenovirus was isolated from livers twice and a reovirus from the intestines once. Supplementation with vitamin E and selenium seemed to improve the situation. The most likely diagnosis is lipidosis, a metabolic disorder with complex etiology, which has rarely been described in turkeys.

A total of 256 fecal specimens were randomly collected from farmed poultry in Germany and screened for the presence of Cryptosporidium spp. by PCR and further characterized by direct automated DNA sequencing. Using a nested PCR amplifying approximately 830 bp 18S rDNA fragment, 7.03% (n = 18) of the samples were Cryptosporidium-positive. In detail, Cryptosporidium was detected in 9.3% (8/86) of turkeys, 5.7% (9/158) of broilers and 8.3% (1/12) of layers. After DNA sequencing, Cryptosporidium parvum the most frequently observed species was identified in 5.1% (13/256) of all poultry species, including 8.1% (7/86) of turkeys, 3.2% (5/158) of broilers and 8.3% (1/12) of layers. Cryptosporidium baileyi was detected in 1.3% (2/256) of the broilers only. Three novel unclassified Cryptosporidium spp. were detected in 1.2% (1/86) of turkeys and 1.3% (2/158) of broilers. The infection rate was high in 13–20 week old turkeys, 1–6 weeks old broilers and >20 weeks old layers but differences between age groups were not significant. This is the first study in Germany uses molecular methods for the detection of Cryptosporidium in poultry. The results indicate that Cryptosporidium parasites are common among broilers and turkeys in Germany. Considering the large size of the poultry industry, the large amount of poultry meat that is consumed and the fact that C. parvum is also the most common Cryptosporidium parasite in humans, poultry might also be a source of human infections.

Serologic tests are a valuable tool for retrospective surveillance of avian influenza viruses (AIV) and monitoring of postvaccination host immune response. Yet collection of serum samples, particularly in adult breeder chickens, is laborious, intrusive to birds, and may pose a serious risk to the biosecurity of a flock. In this study we compared the level of AIV-specific antibody titers in eggs and serum samples obtained from broiler breeder chickens vaccinated at 6, 12, and 18 wk of age with H5N2-inactivated vaccine. Nucleocapsid protein-specific ELISA and hemagglutination inhibition test (HI) against homologous as well as heterologous antigens were used. The eggs and sera were collected at 22, 30, 45, and 50 wk of age (i.e., 4, 12, 27, and 32 wk after the third and final immunization, respectively). Using ELISA, the number of positive egg yolk samples decreased over time after vaccination, from 97% to 47%, while the seropositivity rate of serum samples was 97%-100% during the whole investigation period. No antibody titers were detected in egg white. By HI, antibody titers in serum samples were higher than in egg yolk samples. Compared to the homologous H5N2 antigen, significantly lower HI titers were obtained by using a heterologous H5N1 virus of clade 2.2.1.2. In addition, no HI titers were detected in egg yolk and/or serum samples tested against the antigen of an Egyptian H5N1 antigenic drift variant of clade 2.2.1.1. This study indicates that egg yolk may be used to monitor the postvaccination immune status of broiler breeder chickens and retrospective serosurveillance-by HI when a matching antigen is available as well as by ELISA-particularly for up to 12 wk postvaccination.

The mycoplasma strain ST 57T was isolated from the trachea of a clinically healthy, free-ranging white stork nestling in Nielitz, Mecklenburg-Western Pomerania, Germany. Strain ST 57T grew in fried-egg-shaped colonies on mycoplasma (SP4) agar plates and was dependent on sterol for growth. The organism fermented glucose and did not hydrolyse arginine or urea. The optimal growth temperature was 37 °C, with a temperature range from 23 to 44 °C. Strain ST 57Tcould not be identified as a representative of any of the currently described mycoplasma species by alignment of the 16S rRNA gene sequence or 16S-23S intergenic transcribed spacer region, or by immunobinding assays. Thus, this organism appears to be a representative of a novel species, for which the name Mycoplasma ciconiae sp. nov. is proposed. The type strain is ST 57T (=ATCC BAA-2401T=DSM 25251T). Four further strains of this species are included in this description (ST 24=DSM 29908, ST 56 Clone 1=DSM 29054, ST 99=DSM 29909, ST 102=DSM 29010). The prevalence of this mycoplasma species in clinically healthy, white stork nestlings in northern Germany was determined. Our species-specific PCR detected 57.8 % (48/83) of the samples positive for M. ciconiae sp. nov. As this species appears to be widespread in the healthy free-ranging white stork population, we conclude that this species is either apathogenic or an opportunistic pathogen in white storks.

Ornithobacterium rhinotracheale (ORT) is a gram-negative staining rod. In chickens and turkeys ORT causes a respiratory disease. Between 2009 and 2011 some 714 dry swabs taken from diseased turkeys, broilers, broiler breeders, layers, or from unknown origin were investigated by PCR for the presence of ORT. Swabs that tested positive numbered 197 out of 481 from turkeys (41.0%), 10 out of 144 from broilers or broiler breeders (6.9%), 17 out of 28 from layers (60.7%), and 26 out of 61 from unknown origin (42.6%). The results of three swabs from turkeys were suspect. Furthermore, 310 isolates from turkeys and 62 isolates from unknown origin were typed using an agar gel precipitation (AGP) test. Of the isolates from turkeys, 56.1% belonged to serotype A and 20.6% to serotype E. The prevalence of other isolates was below 10%. Serotypes D, F, and K were not detected. Eleven isolates were not typable with reference sera against serotypes A-L. The three serotypes most often found in the isolates from unknown origin were A (35.5%), B (19.4%), and C (12.9%). The prevalence of other isolates was below 10%. Serotypes F and K were not detected. Seven isolates were not typable with reference sera A-L. Cross-reactions, especially of serotype A isolates with serotypes I, H, and J, were common. Additionally, the partial 16S ribosomal RNA (rRNA) and the complete Or01 genes of reference strains A-H and of nine field isolates were cloned and sequenced. Identity scores of 16S rRNA fragments were between 98% and 100%. Identities of the Or01 sequences were between 94% and 100%. Phylogenetic trees of both genes showed similarities. However, there was no apparent correlation between reference strains and isolates belonging to one serotype, so sequencing of 16S rRNA or of the Or01 gene does not seem to be a suitable method to replace the AGP for serotyping. Further investigations are necessary to clarify the cross-reactions between different serotypes and their real role in the pathogenicity and in consideration of vaccine production.

In recent years, a number of studies about Histomonas meleagridis, and more specifically about experiments in vivo involving H. meleagridis to investigate the pathogenicity and efficacy of drugs or vaccines, have been published. Together with older publications, a considerable amount of information about experimental infections with H. meleagridis exist, which is helpful for planning future animal studies and can reduce the number of birds used in such studies toward better animal welfare. One hundred sixty-seven publications describing experimental infections with H. meleagridis were published in scientific journals between 1920 and 2012. One hundred forty-two of these publications describe infections of turkeys (Meleagris gallopavo) and 52 infections of chickens (Gallus gallus). In 18 studies, experiments involving other species were done. The most popular routes of infection were the intracloacal application of histomonal trophozoites from culture material, from lesions or from feces of infected birds, or using larvae of the cecal worm Heterakis gallinarum (83 studies) and the oral application of eggs or other stages of the cecal worm containing histomonal stages (83 studies). During the last 10 years, intracloacal application of trophozoites has become the most popular way to experimentally infect birds with H. meleagridis due to its high reproducibility and reliability. In most studies, infection doses of several 10,000 or 100,000 histomonal trophozoites were used for infection, and the resulting mortality in turkeys was more than 70 %. First mortality can occur as early as 6 days p.i.; peak mortality usually is 13-15 days p.i. Lower infection doses may delay mortality about 2 days. In chickens infected by the intracloacal route, mortality and clinical signs are rare, but infection rates are similar. Cecal lesions can be observed from 3 to 4 days p.i., lesions up to 3 weeks p.i.; liver lesions may be lacking completely or be present only in a small number of birds. In most studies infecting birds with Heterakis eggs containing histomonal stages, several 100 to 1,000 Heterakis eggs were used. However, lower doses might be sufficient, as infection with as few as 58 eggs per bird caused a mortality up to 90 % in turkeys. Clinical symptoms start 9 days p.i., and first mortality occurs after 12 days, while most of the infected birds die between 19 and 21 days p.i. The infectivity of Heterakis eggs containing histomonal stages for chickens is similar as for turkeys, but mortality and clinical signs are rare. Further infection was done by oral application of histomonal trophozoites either grown in culture or using lesions or feces of infected birds (26 studies). These yielded very mixed results, with infection rates between 0 and more than 80 % in turkeys and chickens. After successful oral infection of turkeys, mortality occurs at roughly the same time as after intracloacal infection. Further 18 studies employed seeder birds to infect in-contact birds. Other means of infection were exposure to contaminated soil or litter (22 studies), feeding contaminated earthworms (7 studies), intracecal inoculation (4 studies), or parenteral injection (4 studies). Main methods to assess the course of the infection were mortality, observation of clinical signs and pathological lesions, monitoring of the weight of the infected birds, and detection of the parasite by various methods.

Highly pathogenic avian influenza virus (HPAIV) of subtype H5N1 causes a devastating disease in poultry but when it accidentally infects humans it can cause death. Therefore, decrease the incidence of H5N1 in humans needs to focus on prevention and control of poultry infections. Conventional control strategies in poultry based on surveillance, stamping out, movement restriction and enforcement of biosecurity measures did not prevent the virus spreading, particularly in developing countries. Several challenges limit efficiency of the vaccines to prevent outbreaks of HPAIV H5N1 in endemic countries. Alternative and complementary approaches to reduce the current burden of H5N1 epidemics in poultry should be encouraged. The use of antiviral chemotherapy and natural compounds, avian-cytokines, RNA interference, genetic breeding and/or development of transgenic poultry warrant further evaluation as integrated intervention strategies for control of HPAIV H5N1 in poultry.

Fowl glioma-inducing virus (FGV), which belongs to avian leukosis virus (ALV) subgroup A, induces fowl glioma. This disease is characterized by multiple nodular gliomatous growths of astrocytes and has been previously reported in Europe, South Africa, Australia, the United States and Japan. FGV and FGV variants have spread to ornamental Japanese fowl, including Japanese bantams (Gallus gallus domesticus), in Japan. However, it is unclear how and where FGV emerged and whether FGV is related to the past fowl glioma in European countries. In this study, the prevalence of FGV in European, Asian and Japanese native chickens was examined. FGV could not be isolated from any chickens in Germany and Asian countries other than Japan. Eighty (26%) out of 307 chickens reared in Japan were positive by FGV-screening nested polymerase chain reaction and 11 FGV variants with an FGV-specific sequence in their 3' untranslated region were isolated. In addition, four other ALVs lacking the FGV-specific sequence were isolated from Japanese bantams with fowl glioma and/or cerebellar hypoplasia. These isolates were considered to be distinct recombinant viruses between FGV variants and endogenous/exogenous avian retroviruses. These results suggest that the variants as well as distinct recombinant ALVs are prevalent among Japanese native chickens in Japan and that FGV may have emerged by recombination among avian retroviruses in the chickens of this country.

This study describes the first isolation of H9N2 avian influenza virus (AIV) from commercial bobwhite quail (Colinus virginianus) in Egypt. Infected birds showed neither clinical signs nor mortality. Virus isolation and real-time reverse transcription polymerase chain reaction confirmed the presence of the H9N2 virus in cloacal swab samples collected at 35 days of age and the absence of other AIV subtypes, including H5 and H7. The hemagglutinin and neuraminidase genes of the isolated virus showed 99.1% and 98.2% nucleotide identity and 97.3% and 100% amino acid identity, respectively, to those of H9N2 viruses currently circulating in poultry in the Middle East. Phylogenetically, the Egyptian H9N2 virus was closely related to viruses of the G1-like lineage isolated from neighbouring countries, indicating possible epidemiological links.

There are only a few reports about the occurrence of coccidia in peafowl and no reports about the occurrence of Eimeria spp. in peafowl kept in Europe. Here, we describe the occurrence of Eimeria pavonina in diseased peafowl from Germany. In January 2011, one young peacock kept in an aviary showed a marked depression. No parasites were detected in samples from the diseased bird, but in samples of birds from the same and other aviaries, coccidian counts were between 400/g and 66,000/g. All peacocks were treated with toltrazuril. After treatment, the clinical condition of the diseased bird improved but, two weeks afterwards, other birds in the aviary were still shedding coccidia in their feces. Based on morphology, the coccidia were identified as E. pavonina. Parts of the 18s rRNA gene and the cytochrome oxidase subunit 1 (cox-1) gene were sequenced. A phylogenetic tree based on the 18s rRNA sequence placed the Eimeria sp. from peafowl closest to Eimeria spp. found in pheasants and partridges as well as to Eimeria meleagrimitis. A phylogenetic tree based on the sequence of cox-1 in contrast suggested a closer relationship to Eimeria necatrix and Eimeria tenella.

SUMMARYLivestock-associated methicillin-resistantStaphylococcus aureus(LA-MRSA) have been isolated from a number of livestock species and persons involved in animal production. We investigated the prevalence of LA-MRSA in fattening turkeys and people living on farms that house fattening turkeys. Eighteen (90%) of 20 investigated flocks were positive for MRSA, and on 12 of the farms 22 (37·3%) of 59 persons sampled were positive for MRSA. People with frequent access to the stables were more likely to be positive for MRSA. In most flocks MRSA that could be assigned to clonal complex (CC) 398 were detected. In five flocks MRSA ofspa-type t002 that is not related to CC398 were identified. Moreover, other methicillin-resistantStaphylococcusspp. were detected on 11 farms and in eight people working on the farms.

Two turkey flocks (male and female) and the environment of their house were investigated for the presence of thermophilic Campylobacter. Sample DNA was extracted directly from fecal material and environmental samples. Bacterial identification was done using a modified Campylobacter species specific multiplex PCR. The times needed for colonization and prevalence in male and female turkeys were determined independently. All environmental samples collected before restocking were negative in the PCR analysis, showing a good hygiene and biosecurity system. The first positive PCR results were obtained in drinking water samples at 6 d of age. Colonization occurred between the second and third week of age, starting in female birds and then followed by the males. Campylobacter jejuni was detected by multiplex PCR at first; later on, Campylobacter coli and mixtures of both were seen. After the 9 wk of age, the colonization of the flocks was completed. Great attention should be given to drinking water as a supposed source of Campylobacter contamination. Multiplex PCR proved to be a rapid, sensitive, and cheap tool for the diagnosis of Campylobacter contamination.

Turkey rotaviruses are one of the major pathogens responsible for the poult enteritis syndrome (PES). In this study a one step real-time reverse transcription polymerase chain reaction (RT-qPCR) assay targeting the rotaviral non-structural protein 4 (NSP4) was developed. The NSP4 is a highly conserved gene inside the turkey rotavirus genome and contains an internal control system to monitor any potential RT-qPCR inhibitors. The detection limit of the optimized NSP4-RT-qPCR assay ranged from 8.15 to 8.15 × 105 copy numbers. In total 149 faecal samples were collected from eight different flocks of commercial turkey farms. Faecal samples from hens and toms were collected separately at 2-week intervals from the 2nd week of age through the 16th and 20th week of age (age of slaughter for female and male, respectively) and tested. One farm reared only hens. The samples were tested previously using conventional RT-PCR targeting the same gene. When the conventional RT-PCR was compared with the developed NSP4-RT-qPCR, the results revealed that 11% of the samples of the conventional RT-PCR were false negative. The results indicate that this NSP4-RT-qPCR is highly sensitive for the detection of turkey rotaviruses in faeces. In addition, it could be suitable for the development of high-throughput screening.

Circulation of an antigenically variant lineage of highly pathogenic avian influenza (HPAI) H5N1 virus in chicken breeder flocks in Egypt is a continuing problem. The protective efficacy of multiple repeated vaccinations using the currently available H5N2 vaccines is unclear. Here, broiler breeder chickens were vaccinated at weeks 6, 12 and 18 with an inactivated H5N2 commercial vaccine. HI-titer against an Egyptian H5N1 field isolate of classic clade 2.2.1 (EGYcls/H5N1) were significantly lower after the first immunization but increased after booster vaccinations. In contrast, no HI titers were induced against an antigenically distinct field virus of the variant lineage of clade 2.2.1 (EGYvar/H5N1). Upon challenge at week 50 mild, if any, clinical signs were observed in the group infected with EGYcls/H5N1 although one of eight (12.5%) birds died. Mortality reached 6/8 (75%) in the EGYvar/H5N1 challenge group. Virus excretion in all vaccinated groups was reduced in amplitude, but in vaccinated surviving birds, time of virus excretion was extended to up to ten days. Strikingly, challenged vaccinated birds kept laying eggs almost throughout the observation period. Virus was detected on the outer egg-shell of 17 of 40 eggs. The majority of the infected eggs were derived from the EGYcls/H5N1 challenged animals; here the virus was detected also in the yolk and albumin. Repeated vaccination using a commercial H5N2-based vaccine broadened the antigen profile of induced antibodies but did not provide adequate protection against heterologous virus variant. In addition, the observation of AIV contaminated eggs from infected flocks highlights the risk of silent virus spread by vaccinated animals and point to eggs as a possible vector.

The poultry populations of Egypt are endemically infected by highly pathogenic avian influenza viruses (HPAIV) of subtype H5N1. Vaccination was chosen as an auxiliary tool to control HPAIV in poultry. Potency of commercial vaccines regarding emerging variants is under discussion. In the current study efficacy of four different inactivated whole H5 virus vaccines representing different sublineages of HPAIV H5N1 were tested in chickens against challenge viruses currently co-circulating in Egypt and representing two antigenically widely distinct HPAIV H5N1 lineages, i.e., "variant" (clade 2.2.1var) and "proper" (clade 2.2.1pro) viruses. All vaccines induced clinical protection against challenge with 2.2.1pro Egyptian strains. In contrast, when challenged with a variant strain, only chickens vaccinated with the homologous Egyptian clade 2.2.1var virus or an inactivated re-assorted H5N1 strain (Re-5, clade 2.3) were protected. However, only the homologous virus induced sterile immunity whereas chickens clinically protected after Re-5 vaccination shed virus at day two after infection indistinguishable to H5N2 vaccines. In conclusion, monitoring vaccine-driven evolution of HPAIV H5N1 by surveillance, antigenic characterization, and challenge studies is essential to assess efficacy of AIV vaccination campaigns.

The efforts exerted to prevent circulation of highly pathogenic avian influenza (HPAI) H5N1 virus in birds are the best way to prevent the emergence of a new virus subtype with pandemic potential. Despite the blanket vaccination strategy against HPAI H5N1 in Egypt, continuous circulation of the virus in poultry has increased since late 2007 as a result of the presence of genetic and antigenic distinct variant strains that have escaped during the immune response of vaccinated birds. Although the suspected poultry flocks have had signs and lesions commonly seen in HPAI H5N1-infected birds, escape of variant strains from detection by real-time reverse transcriptase-PCR (RRT-PCR) was observed. Sequence analysis of these variants revealed multiple single nucleotide substitutions in the primers and probe target sequences of the H5 gene by real-time RT-PCR. This study describes the results of RRT-PCR, modified from an existing protocol with regard to the detection of the partial H5 gene segment of the Egyptian H5N1 divergent viruses and applied to nationwide surveillance. The modified RRT-PCR assay was more sensitive than the original one in the detection of Egyptian isolates, with 104% amplification efficiency. Sixty-one field samples were found to be positive in our assay, but only 51 samples tested positive by the original protocol and were more sensitive than matrix gene RRT-PCR detection assay. A detection limit of 10 mean embryo infective dose (EID50) with the updated oligonucleotides primers and probe set was found. For the foreseeable future, mutation of H5N1 viruses and the endemic situation in developing countries require continuous improvement of current diagnostics to aid in the containment of the H5N1 virus in poultry sectors and to lower the threat of influenza virus spread.

A novel Sarcocystis species has recently been reported in the domestic pigeon (Columba livia f. domestica) as intermediate host, causing severe central nervous signs similar to Paramyxovirus-1 or Salmonella Typhimurium var. cop. infection. Transmission of the parasite via the northern goshawk (Accipiter gentilis) as definitive host has been established. Experimental infection of domestic pigeons with sporocysts excreted by experimentally infected northern goshawks reproduced the natural infection in the pigeon, proving the causative role of the parasite in the disease. Here, we describe in greater detail the course of the fulminant biphasic disease depending on the infectious dose. Pigeons infected with 10(3) or 10(4) sporocysts showed clinical signs of polyuria and apathy around 10-11 days postinfection (dpi) and sudden neurological signs 51-57 dpi as a second phase of disease. Pigeons infected with higher doses died within 7-12 dpi, also showing polyuria and apathy but without nervous signs. At necropsy, livers and spleens had multifocal necroses and infestations with parasitic stages, namely, schizonts. Moreover, lesions and schizonts were also found in the lung, bone marrow, and next to blood vessels in the connective tissue of various organs. Pigeons infected with 102 sporocysts remained symptomless until 58-65 dpi, when sudden central nervous signs occurred. Major histopathologic findings of pigeons with neurological signs were encephalitis and myositis of virtually every skeletal muscle with high infestations of sarcocysts. Only mild myocarditis and very few cysts were found in the heart muscles. Importantly, a sentinel pigeon developed identical lesions when compared to those of low-dose infected pigeons, suggesting a risk of mechanical transmission of sporocysts from freshly infected to uninfected pigeons in a flock. By contrast, chickens failed to develop any clinical signs or pathologic lesions in the same experiment. The findings further characterize the new highly pathogenic disease in domestic pigeons, which clinically mimics paramyxovirosis and salmonellosis in both phases of the disease and exclude chickens as further intermediate host species.

Aspergillosis is an important cause of morbidity and mortality in birds. Turkey poults are known to be particularly susceptible to fungal infection. Although the respiratory tract is the most commonly affected, dissemination can occur into virtually any organ. Here, we report an unusual outbreak of articular aspergillosis in a flock of meat turkeys with clinical signs of lameness. Between 7 and 11 weeks of age, turkeys had severe granulomatous osteoarthritis of the hip joints with necrosis of the femur head. Fungal morphology and PCR amplification and sequencing of the first ITS1-5.8S-ITS2 rDNA region identified Aspergillus fumigatus as the infectious agent. Concurrently, Staphylococcus spp. was isolated from the hip joints, which may have promoted the tropism of the fungus. Mild respiratory tract aspergillosis was observed in only one case. The findings suggest that fungal arthritis may present a specific disease entity in turkeys and should be considered as further cause of lameness in turkeys.

Paromomycin is an aminoglycoside antibiotic with activity against protozoa. Currently, paromomycin is registered for food producing animal species. In a pilot study we evaluated the efficacy of different doses of paromomycin in the feed against Histomonas meleagridis in experimentally challenged turkey poults. Groups consisting of 30 birds each were given feed with 100, 200 and 400 ppm paromomycin, respectively, starting on day 1 through to day 42. One group of 30 birds was left untreated. On day 21 all birds were infected intracloacally with H. meleagridis. Additionally, 10 birds were kept as a non-infected and non-treated control group. Before the challenge there was no significant difference between untreated and treated groups with regards to feed consumption and feed conversion rate. After the challenge, mortality was 80% in the infected nontreated birds. In the treated groups the mortality rate was 73.3%, 43.3% and 20%, respectively. No histomonal DNA was found in caeca and livers of the surviving birds. In addition, higher doses of paromomycin (200 and 400 ppm) led to reduced counts of Clostridium perfringens in the droppings. In conclusion, a prophylactic application of paromomycin as a feed additive was effective against the challenge with H. meleagridis under experimental conditions.

In the present investigation, three turkey lines, namely wild Canadian turkeys (WCT), British United turkey (BUT-Big6) and Kelly-Bronze turkeys (KBT) were compared for their susceptibility to infection with Histomonas meleagridis. All birds were kept on wood shaving as litter from day 1 on during the entire observation period. On day 28, 18-20 birds per turkey line were infected with H. meleagridis intracloacally. All birds were observed for 4 weeks after infection. The mortality rate was 95% in WCT, 78% in BUT-Big6 and 75% in KBT. In WCT, the first deaths occurred at day 6 and ended at day 13 post-infection, whilst for BUT-Big6 and KBT, birds died from days 10 to day 20. In KBT group, the mortality started at day 10 and lasted until day 17 after infection. At necropsy, all birds that died showed lesions typical for histomoniasis in the caeca and liver. The obtained results demonstrate that all tested turkey lines are susceptible to infection; however, the mortality rate for the wild Canadian turkey is statistically significantly higher compared to the other tested two lines.

Mycoplasmas are well-known avian pathogens of poultry and some passerines. Although reported in birds of prey, their role as pathogens is still unclear. Healthy, free-ranging raptor nestlings sampled during a routine ringing (banding) program, and birds of prey from rehabilitation centers, tested positive for Mycoplasma spp. by culture and a genus-specific polymerase chain reaction (PCR). Given the lack of clinical signs and disease, we suggest that mycoplasmas in raptors may be commensal rather than pathogenic. Using immunobinding assay and species-specific PCR tests, Mycoplasma buteonis, M. falconis, and M. gypis were identified; M. falconis was only detected in falcons. Additionally, some isolates could not be identified. This is the first report of Mycoplasma spp. isolations from Western Marsh Harriers (Circus aeroginosus), a Eurasian Hobby (Falco subbuteo), and a Barn Owl (Tyto alba).

To determine reference values for plasma alpha-tocopherol concentrations in falcons and to investigate a possible dietary impact, 29 falcons were randomly selected from a collection of 500 captive breeding falcons fed with 4-week-old chicks. Blood samples collected from each falcon were analyzed for plasma alpha-tocopherol concentrations. Reference values were 16.7–46.1 mg/L with a mean plasma alpha-tocopherol concentration of 27.5 ± 8.7 mg/L. A group of 5 gyr-saker falcon hybrids (Falco rusticolus × Falco cherrug) were fed 1-day-old chicks for 4 weeks; the resultant mean plasma alpha-tocopherol concentration was 34.9 ± 5.6 mg/L. When the diet was changed to turkey breast meat for 4 weeks, the mean plasma alpha-tocopherol concentration decreased significantly to 13.1 ± 2.6 mg/L. Results appeared to support the influence of diet on plasma alpha-tocopherol concentrations in falcons.

Ornithobacterium rhinotracheale (ORT) is an economically important bacterial pathogen of turkeys and chickens worldwide. Since its first detection, a variety of typing methods have been used to gain basic knowledge about the bacterial population structure, an issue that still needs to be addressed. Serological characterization revealed at least 18 different serotypes (A-R) with ORT of serotype A to be predominate among poultry. This study aimed to establish a multilocus sequence typing (MLST) scheme for ORT that could easily be used by other laboratories and allows for worldwide comparison of sequence data. For this purpose, 87 ORT strains from different poultry hosts, geographical origins, years of isolation and serotypes were included in the analysis to identify correlations. Fourteen different sequence types (ST) were found. The most common ST1 was identified in 40 ORT strains from turkeys and chickens on 4 continents and in 3 different European countries. Together with ST9, both STs represented over three quarters (77%) of ORT strains used in the MLST analysis and included strains of frequently cross-reacting ORT serotypes A, E and I. Nine STs were only represented by one ORT strain and might indicate possible avian host, disease or serotype-specific relationships. In contrast, discrepancies between serotype and phylogenetic relatedness were clearly demonstrated by ORT strains that belonged to identical serotypes but differed in their ST. The overall identified low genetic diversity among strains isolated from turkeys and chickens independent of host and geographical origins suggests that ORT has only recently been introduced into domestic poultry and dispersed worldwide.

Liver samples of psittacine birds with a histological suspicion of an adenovirus infection, confirmed by electron microscopy examination, were subjected to virus isolation attempts using a heterologous cell culture system and a homologous cell culture system in the form of chicken embryo liver cells and psittacine embryo fibroblasts, respectively. Whereas isolation in chicken embryo liver cells failed, virus was isolated successfully in the psittacine embryo fibroblasts cell culture system. Molecular investigations identified the virus as a specific psittacine adenovirus (PsAdV). Additionally, on the basis of the hexon gene sequence data obtained, a real-time polymerase chain reaction (PCR) for specific detection of PsAdV was developed. To ensure an exclusive hybridization with PsAdV, selected primers were located within the variable L1 region of the hexon gene. Furthermore, the specificity of the real-time PCR was confirmed by investigation of a panel of different avian adenoviruses and unrelated DNA viruses. Using this PCR, the threshold cycle values obtained support the propagation of PsAdV in the homologous cell culture system in comparison with the chicken cell culture system. Moreover, the developed PCR represents a reliable method for specific and sensitive detection of PsAdV in clinical samples.

The purpose of this study was to investigate the aetiology of the pinching off syndrome (POS), a generalized feather abnormality affecting free-living nestling of the white-tailed sea eagle (Haliaeetus albicilla) in Europe. For the first time, extensive clinical, haematological, biochemical, virological, bacteriological, nutritional, histopathological, parasitological and electron microscopical examinations were performed on three females and one male suffering from POS. Early and increased cytokeratin formation at the base of regenerating feathers and their follicle was observed in affected birds. Ultrathin sections of the feather papillae revealed an extended stratum transitivum and a compact, thickened keratinized stratum corneum. The transitional cells in POS feathers contained vacuoles often associated with the nucleus. Lipofuscin accumulations in neurons, glial cells and islet cells of the pancreas were found in all examined birds. It was not clear whether there is an association between the occurrence of lipofuscin and POS. No evidence was found to suggest that infectious agents (parasites, bacteria, fungi or viruses), malnutrition or hormonal imbalances are involved in the aetiology of POS in white-tailed sea eagles. It remains unclear whether there is a genetic background of POS.

Mycoplasmas are commensals and pathogens of different avian species, especially poultry and passeriforms. The role of mycoplasmas in raptors has not yet been completely determined, and especially not the possibility of vertical transmission. Therefore 424 raptor eggs were examined for the occurrence of mycoplasmas using culture, and 155 of these eggs with a Mycoplasma genus-specific polymerase chain reaction (PCR) assay. This PCR was tested for its sensitivity and specificity, especially for use in a bird population of unknown mycoplasma status (prevalence and species). The size of the amplified PCR product was large (1013 base pairs) to enable use of the product for species differentiation by sequencing. Culture and PCR yielded only one positive result, in an egg of a Northern Goshawk (Accipiter gentilis). The isolate was identified as Mycoplasma lipofaciens using an immunobinding assay, as well as by sequencing part of its 16S rRNA gene.

Histomoniasis (infectious enterohepatitis, blackhead) is caused by the protozoan parasite Histomonas meleagridis (H. meleagridis). After the ban of all prophylactic and therapeutic drugs in the European Union, histomoniasis is increasingly responsible for considerable economic problems to the poultry industry. The aim of this study was to investigate the effect of a herbal product with extracts from cinnamon, garlic, lemon, and rosemary on H. meleagridis in turkey poults in vivo. For this purpose, 60 two-week-old poults were divided into three groups. Group 1 received the herbal product in the feed six days before infection and in water three days before infection, then in feed and drinking water until the end of the experiment. Groups 2 and 3 were left untreated. At week 3 of age, Groups 1 and 2 were infected intracloacally with H. meleagridis. Three weeks after infection the surviving birds were euthanized and examined for pathological lesions. Mortality was 20% in Group 1 and 50% in Group 2. There were no deaths in Group 3. DNA of histomonads was detected in all examined caeca and livers of the dead birds, but was not detected in any examined organ of the surviving birds of all groups. There was no noticeable difference in the lesion scores of the dead birds between the groups. The surviving birds of all groups did not show lesions post mortem. Since all effective prophylactic and therapeutic drugs against histomoniasis were banned in the EU, under given conditions the investigated herbal product seems to be an effective alternative for the reduction of mortality in turkeys caused by histomoniasis.

Blackhead, also known as enterohepatitis, is caused by a protozoan parasite called Histomonas meleagridis. Clinical symptoms are nonspecific. Until now, diagnosis has been mainly based on postmortem lesions and microscopical and histopathological examination. In many cases, especially in layer flocks, these conventional methods are not sufficient, as the lesions are sometimes not clear. The technique for isolation of histomonads in vitro offers many advantages, but the confirmation of histomonads growing in culture may require a time-consuming procedure of rectal inoculation of culture material into chickens or turkeys. The aim of our investigation was to establish a conventional polymerase chain reaction (PCR), a nested PCR, and a real-time PCR, and to examine their specificity as well as sensitivity in the diagnosis of histomoniasis. The obtained results have shown that the conventional PCR is more sensitive than the real-time PCR. Furthermore, the sensitivity of the PCR can be increased by adding the nested PCR. However, the real-time PCR is more specific.