Herpesvirus and Subsequent Usutu Virus Infection in a Great Grey Owl (Strix nebulosa) at the Ljubljana Zoo, Slovenia.

Herpesvirus (HV) has been known to cause disease in owls, with various clinical signs and outcomes for the last several decades. The HV DNA polymerase gene was detected in oropharyngeal and cloacal swabs of a male great grey owl (Strix nebulosa) in a zoological collection in Ljubljana, Slovenia. In the following 4 months, despite continuous HV detection in swabs, no clinical signs with a clear link to HV disease were observed. Hepatoprotective and immunostimulant therapies applied during this period did not prevent HV shedding. Therefore, peroral antiviral therapy with acyclovir (150 mg/kg q24 h for seven days) was performed, and the owl tested negative at the next sampling and remained negative for the next 8 months. After that, the owl again tested positive for HV presence, and the same protocol with antiviral therapy was performed. After 3 weeks with a negative test for HV presence, without any clinical signs of illness, the owl suddenly died because of Usutu virus (USUV) infection. Among all the owls at the zoo, interestingly, only the HV-positive great grey owl died because of USUV infection. The USUV sequence detected and obtained in this study clusters together with other Europe 2 sequences detected in neighboring countries. Our study shows the potential of acyclovir therapy in the prevention of herpesvirus shedding and, moreover, lowering the possibility for spreading HV to other owls and birds. To the best of our knowledge, this is the first report of HV presence and USUV infection in a great grey owl in Slovenia.

First Report of Marek's Disease Virus in Commercial Turkeys in Slovenia.

Marek's disease (MD), caused by Mardivirus gallidalpha 2 (GaAHV-2), also known as MD virus (MDV), is a lymphoproliferative disease that primarily affects chickens. Recently, MDV has been detected in lymphomatous tumors in turkeys in various countries. Between 2021 and 2023, three cases ranging from no to severe clinical disorders (depression, lameness, and increased mortality) occurred in commercial turkey flocks in Slovenia. In all cases, MDV was detected by PCR in DNA samples extracted from organs developing tumor infiltrations. Sequencing and phylogenetic analysis of the meq gene revealed that the GaAHV-2 detected has molecular features of a very virulent pathotype and genetic similarity with GaAHV-2 detected in chickens in Tunisia. This is the first report of MDV in commercial turkeys in Slovenia.

Antimicrobial and Antibiofilm Effect of Commonly Used Disinfectants on Salmonella Infantis Isolates.

Salmonella enterica subsp. enterica serovar Infantis is the most prevalent serovar in broilers and broiler meat in the European Union. The aim of our study was to test the biofilm formation and antimicrobial effect of disinfectants on genetically characterized S. Infantis isolates from poultry, food, and humans. For the biofilm formation under various temperature conditions (8 °C, 20 °C, and 28 °C) and incubation times (72 h and 168 h), the crystal violet staining method was used. The evaluation of the in vitro antimicrobial effect of Ecocid® S, ethanol, and hydrogen peroxide was determined using the broth microdilution method. The antibiofilm effect of subinhibitory concentration (1/8 MIC) of disinfectants was then tested on S. Infantis 323/19 strain that had the highest biofilm formation potential. Our results showed that the biofilm formation was strain-specific; however, it was higher at 20 °C and prolonged incubation time. Moreover, strains carrying a pESI plasmid showed higher biofilm formation potential. The antibiofilm potential of disinfectants on S. Infantis 323/19 strain at 20 °C was effective after a shorter incubation time. As shown in our study, more effective precautionary measures should be implemented to ensure biofilm prevention and removal in order to control the S. Infantis occurrence.

Novel adenoviruses from captive psittacine birds in Slovenia.

To assess the prevalence of adenoviruses in psittacine birds kept in Slovenia, 258 cloacal swabs were collected from different psittacine species and screened by a nested PCR with degenerate, consensus primers targeting the adenoviral DNA polymerase gene. Forty-two samples were found to be positive. By sequencing, 28 samples from 10 different parrot species were identified as the formerly described siadenovirus, psittacine adenovirus 2 (PsAdV-2). A second siadenovirus, a variant of PsAdV-5 (described earlier from Pacific parrotlet, sun parakeet, cockatiel and budgerigar) was found in seven budgerigars, two cockatiels and an amazon parrot species. A variant of Meyer's parrot adenovirus (aviadenovirus, proposed PsAdV-8) was identified in an African grey parrot and a cockatiel. Two novel atadenoviruses were revealed in cockatiel (PsAdV-9) and rose-ringed parakeet (PsAdV-10). These results support the earlier finding that many PsAdVs can cross the species barrier among psittacines, especially effectively in the case of PsAdV-2.

Detection of Herpesviruses in Wild Bird Casualties in Slovenia.

The complete host range of avian herpesviruses in wild birds is unknown, and information about nucleotide sequences is available only in limited cases. The aim of this study was to detect the presence of herpesviruses in wild birds and to gain more information about their phylogenetic relationship. Oropharyngeal and cloacal swabs from 447 wild birds from 15 different orders presented as wildlife casualties were examined for herpesvirus presence with PCR targeting a fragment of the DNA polymerase gene. Herpesviruses were detected in oropharyngeal and/or cloacal swabs in 34 (7.5%) birds belonging to 11 species from six different avian orders: Accipitriformes, Charadriiformes, Columbiformes, Falconiformes, Passeriformes, and Strigiformes. The results of phylogenetic analysis showed that various herpesviruses sequences are present in the wild bird population. Some herpesviruses are host species-specific, whereas in some cases very similar sequences were detected through different avian orders, which confirms findings that herpesviruses are not always restricted to bird species. It seems that herpesvirus transmission could occur by predation from avian prey, and even by superpredation-for example, large owls, such as the Eurasian eagle owl (Bubo bubo) or Ural owl (Strix uralensis), preying on smaller raptors. This can lead to greater infection exposure and is in line with the fact that raptors were the most infected species group. Nevertheless, the individual or simultaneous detection of herpesviruses in oropharyngeal and cloacal swabs shows that both swab samples should be used for herpesvirus detection in wild birds.

Herpesvirus Infection in a Breeding Population of Two Coexisting Strix Owls.

Birds are a frequent host of a large variety of herpesviruses, and infections in them may go unnoticed or may result in fatal disease. In wild breeding populations of owls, there is very limited information about the presence, impact, and potential transmission of herpesvirus. The herpesvirus partial DNA polymerase gene was detected using polymerase chain reaction in oropharyngeal swabs of 16 out of 170 owls examined that were captured in or near nest boxes. Herpesvirus was detected in Ural owls (Strix uralensis), in both adults and young, but not in tawny owls (Strix aluco). In yellow-necked mice (Apodemus flavicollis), as the main prey of tawny owls and Ural owls in the area, herpesvirus was detected in the organs of 2 out of 40 mice captured at the same locations as the owls. Phylogenetic analysis showed that the herpesvirus sequences detected in the Ural owls differed from the herpesvirus sequences detected in the yellow-necked mice. The results indicate that herpesvirus infection exists in the breeding wild Ural owl population. However, herpesvirus-infected owls did not show any clinical or productivity deviances and, based on a phylogenetic comparison of detected herpesvirus sequences and sequences obtained from Genbank database, it seems that mice and other rodents are not the source of owl infections. The most probable transmission pathway is intraspecific, especially from adults to their chicks, but the origin of herpesvirus in owls remains to be investigated.

Health Status and Stress in Different Categories of Racing Pigeons.

The influence of different stress parameters in racing pigeon flocks, such as the presence of diseases and environmental conditions at the time of the races, were described. A total of 96 racing pigeons from 4 pigeon flocks were examined, and health monitoring was carried out. No helminth eggs and coccidia were found. Trichomonas sp. was confirmed in subclinical form. Paramyxoviruses and avian influenza viruses were not confirmed, but circovirus infections were confirmed in all flocks. Chlamydia psittaci was confirmed in one flock. Blood samples were collected, and HI antibody titers against paramyxoviruses before and 25 days after vaccination were determined. To improve the conditions during racing and the welfare of the pigeons, critical points were studied with regard to stress factors during the active training season. Serum corticosterone levels were measured in the blood serum of four different categories of pigeons from each flock. Corticosterone levels were almost twice as high in pigeons from the category that were active throughout the racing season, including medium- and long-distance racing, compared to the other three categories that were not racing actively. Within five hours of the finish of a race, the average serum corticosterone level was 59.4 nmol/L in the most physically active category. The average serum corticosterone level in this category remained at 37.5 nmol/L one month after the last race.

Detection of Laryngotracheitis Virus in Poultry Flocks with Respiratory Disorders in Slovenia.

Infectious laryngotracheitis (ILT) is an acute, highly contagious infectious disease of the upper respiratory tract in chickens and other poultry species that causes significant economic losses in countries worldwide. Between 2017 and 2019, seven outbreaks of mild to severe respiratory disorders with high suspicion of ILT occurred in commercial and backyard poultry flocks in Slovenia. In all submissions, infection with ILT virus (ILTV) was confirmed by PCR, which is the first report of ILT in Slovenia. Circulating ILT strains were characterized by the sequence and phylogenetic analysis of two fragments of the ICP4 gene. Four strains-three detected in non-vaccinated flocks and one in a flock vaccinated against ILT-were identical or very similar to the chicken embryo-origin live virus vaccines, and the other three were closely related to Russian, Chinese, Australian, and American field strains and to tissue culture origin vaccine strains. As in other diseases, coinfections with other respiratory pathogens in confirmed ILT cases may cause a more severe condition and prolong the course of the disease. In our study, coinfections with Mycoplasma synoviae (7/7 tested flocks), infectious bronchitis virus (5/5 tested flocks), Mycoplasma gallisepticum (4/7 tested flocks), Ornithobacterium rhinotracheale (3/4 tested flocks), and avian pox virus (1/2 tested flocks) were confirmed, indicating the importance of these pathogens in the occurrence of ILT infections.

Monitoring of Unhatched Eggs in Hermann's Tortoise (Testudo hermanni) after Artificial Incubation and Possible Improvements in Hatching.

The causes of embryonic mortality in Hermann's tortoises (Testudo hermanni) during artificial incubation were determined. Total egg failure at the end of the hatching period was investigated. The hatching artefacts represented 19.2% (N = 3557) of all eggs (N = 18,520). The viability rate of incubated eggs was 80.8%. The eggs, i.e., embryos, were sorted according to the cause of unsuccessful hatching and subsequently analyzed. Some of the eggs were divided into two or more groups. Unfertilized eggs were confirmed in 61.0%, infected eggs in 52.5%, and eggs in various stages of desiccation in 19.1%. This group also included mummified embryos. Pseudomonas aeruginosa, Bacillus sp., Purpureocillium lilacinum, and Escherichia coli were frequently confirmed in infected eggs. Embryos were divided into three groups: embryos up to 1.0 cm-group 1 (2.2%), embryos from 1.0 cm to 1.5 cm-group 2 (5.4%) and embryos longer than 1.5 cm-group 3 (7.3%) of all unhatched eggs. Inability of embryos to peck the shell was found in 1.3%. These tortoises died shortly before hatching. Embryos still alive from the group 2 and group 3 were confirmed in 0.7% of cases. Dead and alive deformed embryos and twins were detected in the group 3 in 0.5% and 0.1% of cases, respectively. For successful artificial hatching, it is important to establish fumigation with disinfectants prior to incubation and elimination of eggs with different shapes, eggs with broken shells, and eggs weighted under 10 g. Eggs should be candled before and periodically during artificial incubation, and all unfertilized and dead embryos must be removed. Heartbeat monitor is recommended. Proper temperature and humidity, incubation of "clean" eggs on sterile substrate and control for the presence of mites is essential. Monitoring of the parent tortoises is also necessary.

Safety and Efficacy Profile of Live, Intermediate Plus Vaccine Against Infectious Bursal Disease Based on Strain G6.

Infectious bursal disease (IBD) is an acute, highly contagious, immunosuppressive disease of young chickens that causes considerable economic loss in the poultry industry worldwide. Vaccination with live attenuated vaccines is still the most important method used for the control and prevention of IBD in chickens. Here we present the results of in vitro characterization, as well as efficacy and safety testing of a live, intermediate plus vaccine against IBD based on strain G6. Strain characterization confirmed that G6 strain is an intermediate plus strain, showing a high degree of homology with the existing vaccine strains of the same virulence. Safety studies showed that chickens can be vaccinated from 10 days of age. Onset and duration of immunity in specific pathogen free and maternally derived antibodies (MDA) chickens was proven to be 14 and 35 days after vaccination, respectively. When immunizing MDA-positive chickens, vaccine is capable of breakthrough at a titer of ≤500 ELISA units. The field trial conducted on commercial broilers showed a 95% protection against vvIBDV challenge. Stability of the freeze-dried vaccine after reconstitution was confirmed over a period of 3 h. Overall, IBD G6 vaccine has shown good safety and efficacy profile in accordance with European Pharmacopoeia requirements.

Efficacy of Infectious Bronchitis GI-13 (793B) Vaccine Candidate Tested According to the Current European Union Requirements and for Cross-Protection Against Heterologous QX-Like Challenge.

Infectious bronchitis (IB) is a highly contagious viral disease of chickens, known to cause severe economic losses. Vaccination against IB virus (IBV) is an important control measure against the disease. The objective of the present study was to test Avishield IB GI-13, the vaccine candidate against IBV, strain V-173/11 (GI-13 genotype), according to European Pharmacopoeia (Ph. Eur.) efficacy requirements. Laboratory study on specific-pathogen-free (SPF) chickens showed 100% protection against challenge 10 days after vaccination of 1-7 day-old chickens by three recommended routes. Duration of immunity was shown to be at least 8 weeks after vaccination. Chickens with maternally derived antibodies (MDA) were 100% protected against challenge 21 and 35 days after vaccination. Testing of the vaccine candidate in field conditions on commercial broiler and layer farms showed 80-90% protection against homologous challenge after spray (broilers and layers) or oral (broilers) vaccine administration. Serum antibodies were monitored during the studies, and although good seroconversion was observed in MDA-positive chickens 34 days after vaccination or later, the data from SPF chickens indicate that non-humoral immunity is important in protection against challenge. Neutralizing antibodies in tears were detected, however, their level could not be fully linked with individual protection scores. A cross-protection study showed that administration of the combination of Avishield IB H120 vaccine and Avishield IB GI-13 vaccine candidate at day 1, confers good protection against heterologous QX-like challenge. Stability of the vaccine after reconstitution in 0.2% skimmed milk solution or distilled water at room temperature was confirmed over the period of 3 h. The vaccine candidate fully complied with Ph. Eur. requirements, with very good protection levels, indicating that it can be administered already at 1 day of age by spray at the hatchery or at 7 days of age by drinking water on the farm.

Loop-mediated isothermal amplification: rapid molecular detection of virulence genes associated with avian pathogenic Escherichia coli in poultry.

Infections with pathogenic Escherichia coli can lead to different animal- and human-associated diseases. E. coli infections are common in intensive poultry farming, and important economic losses can be expected during infections with avian pathogenic E. coli (APEC) strains followed by colibacillosis. Loop-mediated isothermal amplification (LAMP) assays were developed for rapid detection of 3 APEC-associated virulence genes: sitA, traT, and ompT. All 3 LAMP assays are shown to be specific, repeatable, and reproducible. High sensitivities of the assays are shown, where as few as 1,000 bacterial cells/mL can be detected in different matrices. On-site applicability of this LAMP method is demonstrated through testing of different sample types, from animal swabs and tissues, and from environmental samples collected from 6 commercial poultry farms. All 3 virulence genes were detected at high rates (above 85%) in samples from layer and broiler chickens with clinical signs and, interestingly, high prevalence of those genes was detected also in samples collected from clinically healthy broiler flock (above 75%) while lower prevalence was observed in remaining 3 clinically healthy chicken flocks (less than 75%). Importantly, these virulence genes were detected in almost all of the air samples from 11 randomly selected poultry houses, indicating air as an important route of E. coli spread. Three LAMP assays that target APEC-associated virulence genes are shown to be sensitive and robust and are therefore applicable for rapid on-site testing of various sample types, from animal swabs to air. This on-site LAMP testing protocol offers rapid diagnostics, with results obtained in <35 min, and it can be applied to other important microorganisms to allow the required prompt measures to be taken.

Occurrence of Bacterial and Viral Pathogens in Common and Noninvasive Diagnostic Sampling from Parrots and Racing Pigeons in Slovenia.

Airborne pathogens can cause infections within parrot (Psittaciformes) and pigeon (Columbiformes) holdings and, in the case of zoonoses, can even spread to humans. Air sampling is a useful, noninvasive method which can enhance the common sampling methods for detection of microorganisms in bird flocks. In this study, fecal and air samples were taken from four parrot holdings. Additionally, cloacal and oropharyngeal swabs as well as air samples were taken from 15 racing pigeon holdings. Parrots were examined for psittacine beak and feather disease virus (PBFDV), proventricular dilatation disease virus (PDDV), adenoviruses (AdVs), avian paramyxovirus type-1 (APMV-1), avian influenza virus (AIV), Chlamydia psittaci (CP), and Mycobacterium avium complex (MAC). MAC and AdVs were detected in three parrot holdings, CP was detected in two parrot holdings, and PBFDV and PDDV were each detected in one parrot holding. Pigeons were examined for the pigeon circovirus (PiCV), AdVs, and CP; PiCV and AdVs were detected in all investigated pigeon holdings and CP was detected in five pigeon holdings.

Ornithobacterium rhinotracheale has neuraminidase activity causing desialylation of chicken and turkey serum and tracheal mucus glycoproteins.

Neuraminidases (sialidases) are virulence factors of several poultry pathogens. Ornithobacterium rhinotracheale is a well known poultry pathogen causing respiratory disease in chickens and turkeys all over the world. We investigated whether O. rhinotracheale has neuraminidase enzymatic activity (NEAC). We tested NEAC in 47 O. rhinotracheale strains isolated from turkeys and chickens in eight countries. All strains showed relatively strong NEAC and considerable levels of NEAC were detected also in "cell-free supernatants" of their pelleted cells. Zymography using neuraminidase-specific chromogenic substrate indicated that a protein with molecular mass of ~40kDa and isoelectric point (pI) of ~8.0 is a putative neuraminidase of O. rhinotracheale. Notably, the genome of the type strain of O. rhinotracheale, DSM 15997 contains a gene (Ornrh_1957) encoding a putative neuraminidase with such Mw (39.5 kDa) and pI (8.5). We sequenced a corresponding genomic region of 20 O. rhinotracheale strains and found five distinct types of the neuraminidase gene (termed nanO) sequences. Most diversified nanO sequence was found in two strains isolated from chickens in Hungary in 1995. Their nanO sequences differ from that of the type strain (LMG 9086(T)) in 27 nucleotides. O. rhinotracheale neuraminidase showed capacity to cleave sialic acid from chicken and turkey glycoproteins. It cleaved sialic acid from SAα(2-6)gal moiety of their serum proteins, including immunoglobulin G (IgG) and transferrin. O. rhinotracheale also desialylated chicken and turkey tracheal mucus glycoprotens with SAα(2-3)gal moieties. This study provides the first evidence that O. rhinotracheale has neuraminidase which can desialylate glycoproteins of its natural hosts.

Uterine heterologous malignant mixed Müllerian tumor in a dwarf rabbit (Oryctolagus cuniculus).

Malignant mixed Müllerian tumor (MMMT) is a rare neoplasm of the female genital system. A case of MMMT in the uterus of an 8-year-old female dwarf rabbit, which died with clinical signs associated with severe acute dyspnea and anorexia, is described. At necropsy, an oval, firm tumor was found in each of the 2 uterine horns. Numerous metastases were scattered throughout the mediastinum, thoracic diaphragm, and all pulmonary lobes. Microscopically, the tumors consisted of a poorly demarcated, unencapsulated neoplasm, composed of closely associated carcinomatous and sarcomatous components and areas of osteosarcomatous differentiation. Metastases were composed entirely of the sarcomatous component with osteosarcomatous differentiation. Immunohistochemically, the neoplastic epithelial component was positive for cytokeratin and negative for α-smooth muscle actin (α-SMA), vimentin, and desmin. The sarcomatous component was diffusely and strongly positive for vimentin, focally positive for α-SMA (<20% of cells positive), and negative for desmin. The neoplasm was diagnosed as a heterologous MMMT with metastases to the lung, mediastinum, and thoracic diaphragm.

Surveillance of influenza A viruses in wild birds in Slovenia from 2006 to 2010.

Within the framework of the surveillance program for the early detection of H5 and H7 subtypes of avian influenza (AI) viruses, samples from 2547 wild birds of different species that were collected between 2006 and 2010 were examined by PCR-based methods. AI viruses of various subtypes were detected in 4.4% of birds from four different orders: Anseriformes, Ciconiiformes, Charadriiformes, and Pelecaniformes. Highly pathogenic avian influenza (HPAI) H5N1 viruses were detected only in 2006. HPAI H5N1 virus was confirmed in 1.9% of birds from four different species. Comparison of nucleotide sequences of the H5N1 hemagglutinin gene indicated that two different HPAI H5N1 viruses from the European-Middle Eastern-African clade 1 had been introduced into Slovenia, despite the relatively short duration of the HPAI outbreak. Low pathogenic avian influenza (LPAI) viruses were detected in 2.5% of birds during a 5-yr period. The subtypes H1, H2, H3, H4, H5, H7N7, H8, H10, H11, and H13N6 were determined in 18 out of 64 cases. The highest prevalence (81%) of LPAI viruses, including the H5 subtype, were found in birds sampled as a part of the "active" surveillance system.

Variation of vlhA gene in Mycoplasma synoviae clones isolated from chickens.

Mycoplasma synoviae synthesizes haemagglutinin VlhA, which cleaves into the N-terminal part, a lipoprotein MSPB, and a C-terminal part MSPA. Previous studies have shown that the 3'-end of the expressed vlhA gene can recombine with vlhA pseudogenes in a process called gene conversion, but there have been no data about diversification of the expressed vlhA gene in M. synoviae populations replicating in chickens. Following intratracheal inoculation with the M. synoviae strain ULB 02/T6, which showed only minor vlhA gene variation prior to inoculation, we investigated temporal changes in MSPB epitopes defined by monoclonal antibodies (mAbs) 3B4 and 50, as well as diversification of the vlhA gene sequence in M. synoviae populations recovered from chicken tracheas. In cultures isolated 8 and 18 days post inoculation (p.i.), most colonies showed variation of MSPB epitopes for mAbs 3B4 and 50. They also changed 3'-end vlhA gene sequences. Further diversity of the vlhA gene occurred in cultures isolated 8 weeks and 5 months p.i. The vlhA gene sequences from isolated cultures shared only 65 to 80% sequence identity with vlhA gene of the inoculated ULB 02/T6 culture. Notably, in most of those cultures their vlhA gene sequences contained stop codons potentially causing premature terminations of translation. Interestingly, in one culture isolated 8 weeks p.i. (clone T6-8W/IT2A) the 3'-vlhA gene sequence was identical in the last 1140 bases to that of the first vlhA pseudogene positioned the most far (upstream) of the expressed vlhA gene. This is the first demonstration of temporal diversity of the vlhA gene in M. synoviae populations isolated from chicken tracheas.

Field efficacy of different vaccines against infectious bursal disease in broiler flocks.

A field study was performed to determine the efficacy of three commercially available vaccines against infectious bursal disease (IBD) in commercial broilers raised in a high IBD virus (IBDV) risk area. Live attenuated intermediate and intermediate plus vaccines were used in four flocks. Birds were vaccinated orally at the estimated vaccination time. Three broiler flocks were vaccinated subcutaneously with a turkey herpesvirus (HVT)-IBD vector vaccine at one day old. Evaluation of the efficacy of different vaccines was focused on humoral immune response, bursa/body weight (B/Bw) ratio, molecular detection of IBDV in ileocaecal tonsils and bursa of Fabricius, and production parameters. The serological results showed that although the uptake of all three vaccine strains was confirmed in the lymphoid organs, no significant antibody response to vaccination was detected in flocks vaccinated with intermediate and intermediate plus vaccines. A significant increase in antibody titres detected in flocks vaccinated with the vector vaccine indicated its ability to induce an immune response in birds with a high level of maternally derived antibodies. Observations obtained in this field trial did not confirm the expected reduction of the B/Bw ratio in flocks vaccinated with less attenuated vaccines. No significant differences were observed between birds vaccinated with the vector vaccine and those immunised with the intermediate plus vaccine. Very virulent IBDV was confirmed in the flock vaccinated with the intermediate vaccine. The infection induced reduced B/Bw and moderate mortality but did not affect the production parameters. Field infection was not detected in broilers vaccinated with the intermediate plus vaccine and the vector vaccine.

Neuraminidase of Mycoplasma synoviae desialylates heavy chain of the chicken immunoglobulin G and glycoproteins of chicken tracheal mucus.

Major poultry pathogens, Mycoplasma gallisepticum and Mycoplasma synoviae share several genes, including nanH that encodes their sialidases (neuraminidases). Previous studies have shown considerable differences in neuraminidase enzymatic activity (NEAC) in M. synoviae strains and NEAC absence in individual cultures of two strains, ULB 925 and ULB 9122. The present study shows that the cultures lacking NEAC did not express NanH neuraminidase detectable by specific antibodies. In cultures of M. synoviae ULB 925 and ULB 9122, which lacked NEAC and detectable NanH, deletions of a single adenine in different nanH regions of each strain created translational frameshifts resulting in TAA (UAA) stop codons and premature termination of translation. ULB 925 and ULB 9122 with such nanH mutations did not desialylate reference fetuin and transferrin or chicken glycoproteins that M. synoviae strains with NEAC efficiently desialylated. They desialylated several chicken serum glycoproteins with SAα(2-6)gal moieties, including the immunoglobulin G heavy chain. Neuraminidase inhibitor 2,3-didehydro-2-deoxy-N-acetylneuraminic acid inhibited such desialylation otherwise caused by M. synoviae WVU 1853 neuraminidase. WVU 1853 also cleaved sialic acid from SAα(2-3)gal moieties from glycoproteins of mucus from chicken tracheas. This is the first demonstration that M. synoviae desialylates glycoproteins of its host.

Possible sources and spreading routes of highly pathogenic avian influenza virus subtype H5N1 infections in poultry and wild birds in Central Europe in 2007 inferred through likelihood analyses.

Recurrent outbreaks of H5N1 HPAIV occurred in several Central European countries in 2007. In-depth phylogenetic analyses which included full-length genomic sequences of the viruses involved were performed to elucidate possible origins of incursions and transmission pathways. Tree reconstructions as well as host-shift and ancestral area inferences were conducted in a maximum likelihood framework. All viruses belonged to a separate subgroup (termed "EMA-3") within clade 2.2, and, thus, were distinct from two lineages of HPAIV H5N1 viruses (termed "EMA-1" and "EMA-2") present in the same geographic area in 2006. Analysis of concatenated coding regions of all eight genome segments significantly improved resolution and robustness of the reconstructed phylogenies as compared to single gene analyses. At the same time, the methodological limits to establish retrospectively transmission networks in a comparatively small geographic region and spanning a short period of time became evident when only few corroborating field-epidemiological data are available. Ambiguities remained concerning the origin of the EMA-3 viruses from a region covering Southeast Germany and the Czech Republic as well as routes of spread to other European countries. AIV monitoring programmes in place for wild birds and poultry in these countries did not reveal presence of these viruses in either population. Host switches between domestic poultry and wild bird populations occurred several times. Analysis of outbreaks in Northeast Germany and nearby Northern Poland in December 2007 demonstrated that geographic and even temporal vicinity of outbreaks does not necessarily indicate a common source of incursion.