Pathogenesis of avian bornavirus in experimentally infected cockatiels.

Avian bornavirus (ABV) is the presumed causative agent of proventricular dilatation disease (PDD), a major fatal disease in psittacines. However, the influencing factors and pathogenesis of PDD are not known and natural ABV infection exhibits remarkable variability. We investigated the course of infection in 18 cockatiels that were intracerebrally and intravenously inoculated with ABV. A persistent ABV infection developed in all 18 cockatiels, but, as in natural infection, clinical disease patterns varied. Over 33 weeks, we simultaneously studied seroconversion, presence of viral RNA and antigens, infectious virus, histopathologic alterations, and clinical signs of infection in the ABV-infected birds. Our study results further confirm the etiologic role of ABV in the development of PDD, and they provide basis for further investigations of the pathogenetic mechanisms and disease-inducing factors for the development of PDD.

Isolation of herpesvirus and Newcastle disease virus from White Storks (Ciconia ciconia) maintained at four rehabilitation centres in northern Germany during 1983 to 2001 and failure to detect antibodies against avian influenza A viruses of subtypes H5 and H7 in these birds.

Herpesvirus isolations from peripheral white blood cells of 253 White Storks (Ciconia ciconia) were obtained during a long-term study (1983 to 2001). The storks lived for a few months to 20 years at four rehabilitation centres. Isolates were obtained from 83 of 253 storks. This herpesvirus is indigenous for storks and unrelated to any other avian herpesvirus. Significantly more herpesvirus isolates were obtained during spring than in autumn samplings. The intervals between the first and last virus isolation ranged from 1 to 15 years. Herpesvirus isolates were simultaneously obtained from white blood cells and from pharyngeal swabs of four of 34 storks but not from cloacal swabs. Neutralizing antibodies to stork herpesvirus were detected in 178 of 191 examined blood plasma samples. Neutralizing antibodies against stork herpesvirus did not correlate with herpesvirus viraemia. The results further substantiate the persistence of herpesvirus in White Storks and underline the previously unrecorded long periods of virus and antibody presence. Virulent avian paramyxovirus type 1 (APMV-1; Newcastle disease virus) was isolated from white blood cells during 1992 and 1993 from four healthy migrating storks, and possessed virulence markers on the cleavage site of the H and F genes. These properties resemble the NE type of APMV-1. Haemagglutination inhibition antibodies against APMV-1 were detected in 16 of 191 blood plasma samples. Avian influenza A virus was not isolated and antibodies against subtypes H5 and H7 were not detected.

Occurrence of avian bornavirus infection in captive psittacines in various European countries and its association with proventricular dilatation disease.

A total of 1442 live birds and 73 dead birds out of 215 bird collections in Spain, Germany, Italy, the UK and Denmark were tested for avian bornavirus (ABV) infection by four different methods. The majority of the birds were psittacines belonging to 54 different genera of the order Psittaciformes. In total, 22.8% of the birds reacted positive for ABV in at least one of the tests. Combined testing of swabs from the crop and cloaca, and serum for the diagnosis of ABV infection in live birds revealed that virus shedding and antibody production coincided in only one-fifth of the positive birds so that the examination of these three samples is recommended for reliable ABV diagnosis. By statistical analysis of this large number of samples, the ABV infection proved to be highly significant (P <0.001) associated with histopathologically confirmed proventricular dilatation disease (PDD) in dead birds as well as with clinically assumed PDD in live birds. However, ABV infection was also detected in psittacines without pathological lesions or clinical signs of PDD. Twelve non-psittacine birds belonging to the genera Aburria, Ciconia, Geopelia, Leucopsar and Pavo were tested negative for ABV infection. Within the order of Psittaciformes, birds belonging to 33 different genera reacted positive for ABV. In 16 of these psittacine genera, the ABV infection was demonstrated for the first time. The present study emphasizes the widespread occurrence of clinically variable ABV infections in Europe by analysing a large number of specimens from a broad range of bird species in several assays.

Indirect immunofluorescence assay for intra vitam diagnosis of avian bornavirus infection in psittacine birds.

Different avian bornavirus (ABV) genotypes have recently been detected in psittacine birds with proventricular dilatation disease (PDD), an inflammatory fatal central and peripheral nervous system disorder. An indirect immunofluorescence assay (IIFA) for intra vitam demonstration of ABV-specific serum antibodies was established since reverse transcription-PCR (RT-PCR) assays may not detect all ABV variants.

[Mortality in free living siskins (Spinus spinus Linnaeus, 1758) due to Salmonella typhimurium, phage type DT104 and DT013]. / Todesfälle bei frei lebenden Zeisigen (Spinus spinus Linnaeus, 1758) durch Salmonella Typhimurium DT104 und DT013.

This report deals with an enzootic due to Salmonella Typhimurium in two free living Eurasian siskins (Spinus spinus Linnaeus, 1758). Other birds in the vicinity of the siskins were not affected. Clinical signs consisted of non-specific symptoms such as ruffled plumage, apathy and reduced food intake. During necropsy, gross lesions were enlarged livers with focal necrosis, pale spleens, enlarged kidneys, pneumonia and enteritis. Salmonella Typhimurium was isolated from internal organs in pure culture. Using the polymerase chain reaction, the detection of Salmonella according to EN ISO 6579:2002 was confirmed. The detailed characterisation of both isolates in the Federal Institute for Risk Assessment and in the Robert Koch Institute yielded for the first siskin Salmonella Typhimurium, 4, 5, 12: i : 1, 2, LT DT104, BT a and for the second siskin Salmonella Typhimurium, 4,12 i : 1, 2, LT DT013, BT c. These phage types were identified for the first time in siskins. The detected phage types have importance as causes of disease not only for free living siskins but also as infectious and zoonotic agents for domestic poultry and poultry products.

Phylogenetic analysis reveals extensive evolution of avian paramyxovirus type 1 strains of pigeons (Columba livia) and suggests multiple species transmission.

Partial sequence and residue substitution analyses of the fusion protein gene were performed for 68 strains of avian paramyxovirus type 1 of pigeons (PPMV-1), an antigenic variant of Newcastle disease virus (NDV) of chickens, derived from 16 countries between 1978 and 2002. The majority of isolates clustered into a single genetic lineage, termed VIb/1, within genotype VI of NDV strains of chickens, whereas a small number of isolates that originated in Croatia after 1995, grouped in a highly diverged lineage, termed VIb/2, indicating a separate host-switching event from that of VIb/1 strains. Four distinct subgroups of lineage VIb/1, Iraqi (IQ), early European (EU/ea), North American (NA) and recent European (EU/re) have emerged and circulated in the past decades. Subgroup EU/ea and NA strains were responsible for the main streams of infection in the 1980s, while EU/re viruses for infections in the 1990s. The degree of genetic diversity of viruses in the early phase of the epizootic suggested a prolonged infection period of the pigeon-type viruses prior to the emergence of the disease in the early 1980s. Shared derived character analysis showed a close genetic relationship to Sudanese viruses from the mid-1970, suggesting that PPMV-1 viruses could be of African origin.

Genetic characterization of a herpesvirus isolate from a superb starling (Lamprotornis superbus) as a psittacid herpesvirus genotype 1.

Four genotypes of the psittacid herpesvirus (PsHV) cause Pacheco disease in parrots. Viruses that are serologically cross-reactive to the PsHVs have also been isolated from passerine species. DNA was amplified from a herpesvirus isolated from a superb starling (Lamprotornis superbus) with PsHV-specific primers and polymerase chain reaction. A comparison of the partial sequence of the UL 16 gene from this herpesvirus with sequences from viruses of known PsHV genotypes showed that the herpesvirus from the superb starling was a PsHV genotype 1 virus. This finding expands the range of birds that are known to be susceptible to PsHV genotype 1 infections and suggests that PsHVs should be considered as a differential in passerines with herpesvirus infections.

Isolation and characterization of avian paramyxovirus type 3b from farmed Namibian ostriches (Struthio camelus f. dom.).

Meat and skin from farmed ostriches are valuable products for European consumers. The EU regulations require that ostrich products deamed for export need to come from ostriches that are free of antibodies against Newcastle disease virus (avian paramxovirus type 1, aPMV-1). After the detection of antibodies against aPMV-1 in one of five ostrich farms in Namibia, attempts were made to isolate the causative virus. No aPMV-1 but an avian paramyxovirus type 3 (aPMV-3) was isolated from five pharyngeal/cloacal swabs of clinically healthy farmed Namibian ostriches. Subtype determination proved that all isolates are members of the subtype aPMV-3 of psittacine bird origin and were designated as aPMV-3b. In the haemagglutination inhibition test, the aPMV-3b isolates cross-reacted with aPMV-1. This allows the conclusion that the antibodies originally detected in sera of the ostriches are due to the cross-reaction with aPMV-3b, rather than to an infection with aPMV-1.To our knowledge, this is the first description of the occurrence of aPMV-3b in farmed ostriches.

A disease complex associated with pigeon circovirus infection, young pigeon disease syndrome.

In order to collect more convincing data on the aetiological agent of young pigeon disease syndrome (YPDS), a comprehensive study was performed on pigeons in German lofts with or without outbreaks of YPDS. The investigations included examination of histories, clinical signs and pathology, as well as parasitological and microbiological analysis. Pigeons in their 4th to 12th week of life exhibited clinical signs at higher frequency and with greater severity than pigeons of other ages. Greenish liquid in the crop, proventriculus and ventriculus, and yellow fluid in the small intestine were seen more often in YPDS-affected pigeons. Escherichia coli and Klebsiella pneumoniae were isolated more frequently from these birds. Depletion of splenic and bursal lymphocytes was only seen in pigeons with YPDS. Inclusion bodies were present in various organs, especially the bursa of Fabricius. The genome of pigeon circovirus was detected in lymphoid tissues from all pigeons with YPDS. The results of this study indicate that YPDS is a multifactorial disease in which pigeon circovirus might be a crucial factor, possibly by inducing immunosuppression in infected birds.

Sequencing of the Chlamydophila psittaci ompA gene reveals a new genotype, E/B, and the need for a rapid discriminatory genotyping method.

Twenty-one avian Chlamydophila psittaci isolates from different European countries were characterized using ompA restriction fragment length polymorphism, ompA sequencing, and major outer membrane protein serotyping. Results reveal the presence of a new genotype, E/B, in several European countries and stress the need for a discriminatory rapid genotyping method.

Molecular phylogeny of the psittacid herpesviruses causing Pacheco's disease: correlation of genotype with phenotypic expression.

Fragments of 419 bp of the UL16 open reading frame from 73 psittacid herpesviruses (PsHVs) from the United States and Europe were sequenced. All viruses caused Pacheco's disease, and serotypes of the European isolates were known. A phylogenetic tree derived from these sequences demonstrated that the PsHVs that cause Pacheco's disease comprised four major genotypes, with each genotype including between two and four variants. With the exception of two viruses, the serotypes of the virus isolates could be predicted by the genotypes. Genotypes 1 and 4 corresponded to serotype 1 isolates, genotype 2 corresponded to serotype 2 isolates, and genotype 3 corresponded to serotype 3 isolates. The single serotype 4 virus mapped to genotype 4. DNA from a virus with a unique serotype could not be amplified with primers that amplified DNA from all other PsHVs, and its classification remains unknown. Viruses representing all four genotypes were found in both the United States and Europe, and it was therefore predicted that serotypes 1, 2, and 3 were present in the United States. Serotype 4 was represented by a single European isolate that could not be genetically distinguished from serotype 1 viruses; therefore, the presence of serotype 4 in the United States could not be predicted. Viruses of genotype 4 were found to be the most commonly associated with Pacheco's disease in macaws and conures and were least likely to be isolated in chicken embryo fibroblasts in the United States. All four genotypes caused deaths in Amazon parrots, but genotype 4 was associated with Pacheco's disease only in Amazons in Europe. Genotypes 2, 3, and 4, but not 1, were found in African grey parrots. Although parrots from the Pacific distribution represent a relatively small percentage of the total number of birds with Pacheco's disease, all four genotypes were found to cause disease in these species.

Shell growth and chamber formation of aquarium-reared Nautilus pompilius (Mollusca, Cephalopoda) by X-ray analysis.

Observations on the growth rate of aquarium maintained Nautilus pompilius in different developmental stages, i.e. juveniles (shell length about 8.75 cm), late juveniles (approximately 10 cm), and early adolescent (approximately 13.5 cm), indicate that this species is fully grown at an age of 7.3-8 years. The age calculations are based on two different computations: (1) the measurement of the increase of the shell length per day and (2) the formation of new septa in time intervals of 150+/-5 days, as demonstrated by X-ray analyses. After N. pompilius hatches, its shell grows about 139 mm to reach full growth and approximately 28 septa are formed. With an increase of the shell length of 0.052 mm per day, it takes about 2,673 days (7.3 years) to reach maturity. Provided that the process of chamber formation follows an exponential function, these computations result in approximately 2,925 days (8 years) to reach full maturity. Supposing that N. pompilius may live for several years after onset of maturity like Nautilus belauensis, the total life span for this species may exceed 11-12 years.

Recombinant expression of a truncated capsid protein of beak and feather disease virus and its application in serological tests.

Beak and feather disease virus (BFDV) causes severe disease characterized by irreversible feather disorders and severe immunosuppression in many psittacine species. BFDV cannot be propagated in tissue or cell cultures, rendering virus propagation and thus diagnosis rather difficult. To develop reliable diagnostic methods, the region encoding the BFDV capsid protein C1 was cloned from an infected sulphur-crested cockatoo (Cacatua galerita). Phylogenetic analysis showed this gene had 76.3 to 83.2% amino acid identity to published sequences. No protein was detected after induction of full-length C1 expression in Escherichia coli. However, deletion of an amino-terminal arginine-rich sequence facilitated expression. C1(39-244)-His, a polyhistidine-tailed variant of this protein, was purified and used for immunization of chickens. The immune sera detected C1 with an apparent molecular weight of 27 kDa in western blots of organ homogenates of BFDV-infected birds. Using C1(39-244)-His as antigen, 11 psittacine sera were tested for the presence of BFDV-specific antibodies by enzyme-linked immunosorbent assay and immunoblotting. The results obtained correlated well with the BFDV-specific haemagglutination inhibition activity of the sera, suggesting C1(39-244)-His has value as a recombinant antigen for BFDV-specific serological tests.

The digestive tract of Nautilus pompilius (Cephalopoda, Tetrabranchiata): an X-ray analytical and computational tomography study on the living animal.

Using X-ray analytical studies and computational tomography, the position of the digestive tract of the tetrabranchiate cephalopod Nautilus pompilius L. was demonstrated in a living animal. For the first time, a detailed analysis of the rate of digestion and the duration of the different phases of a digestive cycle has been made using these in vivo methods. At 20 min after food intake, the food has entered the stomach, where it is reduced to small pieces; most is stored in the crop, which is enlarged to approximately four times its original size. The chyme reaches the midgut gland 3 h and the rectal loop 5 h after food intake. The time between food intake and elimination is 12 h. Thus, in Nautilus pompilius, digestion takes approximately the same time as described for nectobenthic sepioids and benthic octopods but is approximately 6 h longer than in loliginids, which have a pelagic mode of life. Furthermore, the three-dimensional structure of the digestive tract of a living Nautilus pompilius L. was demonstrated using computational tomography.