Ursula Heffels-Redmann
email: ursula.heffels-redmann@vetmed.uni-giessen.de

Dirk Enderlein

Sibylle Herzog

Christiane Herden

Anne Piepenbring

Daniel Neumann

Hermann Müller

Sara Capelli

Helga Gerlach

Erhard F Kaleta

Ursula ; Justus

Justus Liebig

Erhard ; Kaleta

Hermann Müller

Heiner Müller

Kirstin Oberhäuser

Erhard F Kaleta

Michael Lierz

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

Keywords: avian diseases, parrots, avian bornavirus diagnostics, epidemiology

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

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 crop and cloaca, and serum for the diagnosis of ABV infection in alive 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. This 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. [START_REF] Kistler | Recovery of divergent avian bornaviruses from cases of proventricular dilatation disease: identification of a candidate etiologic agent[END_REF][START_REF] Honkavuori | Novel Borna virus in psittacine birds with proventricular dilatation disease[END_REF]. The ABV RNA was detected in parrots which died from proventicular dilation disease (PDD). After these first publications, further reports of ABV infections in PDD affected psittacines originated from Germany [START_REF] Enderlein | Aviäres Bornavirus: Antikörper-und Genomnachweis in PDD positiven Vögeln[END_REF][START_REF] Lierz | Anatomic distribution of avian Borna virus in parrots, its occurrence in clinically healthy birds and ABV antibody detection[END_REF][START_REF] Rinder | Broad tissue and cell tropism of avian bornavirus in parrots with proventricular dilatation disease[END_REF]), Austria, Switzerland, Hungary, Australia (Weissenböck et al., 2009a) and Canada (Raghav et al., 2010). Besides, a report on the detection of ABV genome in a canary (Serinus canaria) with enteric ganglioneuritis and encephalitis indicates that not only psittacine birds may be susceptible for ABV infection (Weissenböck et al., 2009b).

PDD is a progressive, often fatal disease of captive psittacine birds worldwide. The disease is characterized by lymphohistioplasmocytic infiltration of the ganglia of the central and peripheral nerve system leading to gastrointestinal dysfunction and associated wasting as well as neurological symptoms. PDD is one of the most threatening diseases for parrots including endangered psittacine species. It has been found in about 60 species belonging to 20 different psittacine genera [START_REF] Lutz | Psittacine proventricular dilatation syndrome in an Umbrella cockatoo[END_REF][START_REF] Vice | Myocarditis as a component of psittacine proventricular dilatation syndrome in a Patagonian conure[END_REF][START_REF] Gregory | Review of proventricular dilatation syndrome[END_REF][START_REF] Sullivan | First case of psittacine proventricular dilatation syndrome (macaw wasting disease) in Australia[END_REF][START_REF] Berhane | Peripheral neuritis in psittacine birds with proventricular dilatation disease[END_REF][START_REF] Lierz | Proventricular dilatation disease[END_REF][START_REF] Lublin | An outbreak of proventricular dilatation disease in a psittacine breading farm in Israel[END_REF], Lierz et al., 2010), but also in some non-psittacine bird species [START_REF] Staeheli | Avian bornavirus associated with fatal disease in psittacine birds[END_REF]. Since its first description in the late 1970s several viruses (adenovirus, herpesvirus, polyomavirus, eastern and western equine encephalitis viruses, paramyxovirus types 1 and 3) [START_REF] Mannl | Neuropathic gastric dilatation in Psittaciformes[END_REF][START_REF] Sullivan | First case of psittacine proventricular dilatation syndrome (macaw wasting disease) in Australia[END_REF][START_REF] Vice | Myocarditis as a component of psittacine proventricular dilatation syndrome in a Patagonian conure[END_REF][START_REF] Gregory | Proventricular dilatation disease[END_REF][START_REF] Gregory | Experimental transmission of psittacine proventricular dilatation disease (PDD) and preliminary characterization of a virus recovered from birds with naturally occuring and experimentally induced PDD[END_REF]Grund et al., 2002;[START_REF] Gough | Isolation of a coronavirus from a green-checked Amazon parrot (Amazona viridigenalis Cassin)[END_REF][START_REF] Lublin | An outbreak of proventricular dilatation disease in a psittacine breading farm in Israel[END_REF]Orosz & Dahlhausen, 2007) have been proposed to be the aetiologic and Patagonian conures (Cyanoliseus patagonis) provided evidence for the aetiological role of ABV in the development of PDD [START_REF] Gancz | Experimental induction of proventricular dilatation disease in cockatiels (Nymphicus hollandicus) inoculated with brain homogenates containing avian bornavirus 4[END_REF][START_REF] Gray | Use of avian bornavirus isolates to induce proventricular dilatation disease in conures[END_REF]. Besides, ABV infections were also found in psittacines with no clinical signs of PDD [START_REF] De Kloet | Presence of avian bornavirus RNA and anti-avian bornavirus antibodies in apparently healthy macaws[END_REF][START_REF] Lierz | Anatomic distribution of avian Borna virus in parrots, its occurrence in clinically healthy birds and ABV antibody detection[END_REF][START_REF] Villanueva | The diagnosis of proventricular dilatation disease: Use of a Western blot assay to detect antibodies against avian Borna virus[END_REF].

Meanwhile, different diagnostic methods have been developed for the direct and indirect diagnosis of ABV. ABV RNA can be detected by RT-PCR using various primer sets.

In PDD affected dead psittacine birds ABV RNA was demonstated not only in the nervous system and the gastrointestinal tract but in nearly all other tissues [START_REF] Kistler | Recovery of divergent avian bornaviruses from cases of proventricular dilatation disease: identification of a candidate etiologic agent[END_REF][START_REF] Honkavuori | Novel Borna virus in psittacine birds with proventricular dilatation disease[END_REF][START_REF] Lierz | Anatomic distribution of avian Borna virus in parrots, its occurrence in clinically healthy birds and ABV antibody detection[END_REF][START_REF] Rinder | Broad tissue and cell tropism of avian bornavirus in parrots with proventricular dilatation disease[END_REF][START_REF] Villanueva | The diagnosis of proventricular dilatation disease: Use of a Western blot assay to detect antibodies against avian Borna virus[END_REF][START_REF] Enderlein | Aviäres Bornavirus: Antikörper-und Genomnachweis in PDD positiven Vögeln[END_REF][START_REF] Gray | Use of avian bornavirus isolates to induce proventricular dilatation disease in conures[END_REF][START_REF] Kistler | Analysis of naturally occuring avian bornavirus infection and transmission during an outbreak of proventricular dilatation disease among captive psittacine birds[END_REF]Raghav et al., 2010). Further methods for the direct detection of ABV in dead birds as well as in crop biopsies are immunohistochemistry [START_REF] Rinder | Broad tissue and cell tropism of avian bornavirus in parrots with proventricular dilatation disease[END_REF]Ouyang et al., 2009;Weissenböck et al., 2009a;[START_REF] Herzog | Indirect immunofluorescence assay suitable for intra vitam diagnosis of avian bornavirus infection in psittacine birds[END_REF]Raghav et al., 2010), virus isolation in the quail cell lines CEC-32 and QM7 [START_REF] Rinder | Broad tissue and cell tropism of avian bornavirus in parrots with proventricular dilatation disease[END_REF], Herzog et al., 2010) or duck embryo fibroblasts [START_REF] Gray | Use of avian bornavirus isolates to induce proventricular dilatation disease in conures[END_REF], and in situ hybridization [START_REF] Weissenböck | Localization of avian bornavirus RNA by in situ hybridization in tissues of psittacine birds with proventricular dilatation disease[END_REF]. In live birds, ABV can be diagnosed directly by detection of ABV RNA using RT-PCR in faeces, swabs of crop and cloaca, and blood [START_REF] Rinder | Broad tissue and cell tropism of avian bornavirus in parrots with proventricular dilatation disease[END_REF][START_REF] Lierz | Anatomic distribution of avian Borna virus in parrots, its occurrence in clinically healthy birds and ABV antibody detection[END_REF][START_REF] Enderlein | Aviäres Bornavirus: Antikörper-und Genomnachweis in PDD positiven Vögeln[END_REF][START_REF] Gray | Use of avian bornavirus isolates to induce proventricular dilatation disease in conures[END_REF][START_REF] Kistler | Analysis of naturally occuring avian bornavirus infection and transmission during an outbreak of proventricular dilatation disease among captive psittacine birds[END_REF]. As serological methods for the demonstration of of anti-ABV antibodies a Western blot assay [START_REF] De Kloet | Presence of avian bornavirus RNA and anti-avian bornavirus antibodies in apparently healthy macaws[END_REF][START_REF] Lierz | Anatomic distribution of avian Borna virus in parrots, its occurrence in clinically healthy birds and ABV antibody detection[END_REF][START_REF] Villanueva | The diagnosis of proventricular dilatation disease: Use of a Western blot assay to detect antibodies against avian Borna virus[END_REF], an ELISA [START_REF] De Kloet | Presence of avian bornavirus RNA and anti-avian bornavirus antibodies in apparently healthy macaws[END_REF] and an indirect immunofluorescence assay [START_REF] Gray | Use of avian bornavirus isolates to induce proventricular dilatation disease in conures[END_REF][START_REF] Herzog | Indirect immunofluorescence assay suitable for intra vitam diagnosis of avian bornavirus infection in psittacine birds[END_REF] have been developed.

In this study, analysis of the results of testing a high number of samples from psittacine and non-psittacine birds from Spain, Germany, Italy, United Kingdom and

Materials and Methods

Birds and samples. In total, 1,515 birds out of 215 different bird collections in Spain, Germany, Italy, United Kingdom and Denmark were included in the study (Table 1). The birds or different samples of them had been submitted by veterinarians, bird owners and breeders as well as zoos for diagnostic purposes in most cases without any or with poor anamnestic data concerning number and species of other birds in the flock. The examined birds belonged to 54 different genera of the order Psittaciformes and 5 genera of nonpsittacine birds (Table 2). Seventy three of these birds with or without clinical suspicion of PDD were dead birds (42) or the organs of dead birds which were submitted freshly (20) or fixed in formalin or embedded in paraffin (11). From 1,442 live birds, sera (1,349), crop (347) and cloacal (381) swabs were received as either single or combined samples per bird. Sixty three of these live birds were suspected of PDD due to clinical signs like undigested feed in the faeces, loss of weight, proventricular dilatation in radiography and/or central nervous symptoms. From the other live birds, no clinical signs were reported, and samples of them were taken to detect subclinical ABV infections.

General examination. All dead birds were necropsied, and the brain, spinal cord, retina, Nervus ischiadicus, crop, proventriculus, gizzard, small and large intestine, heart, liver, kidney, pancreas, pectoral muscle, skin were collected. These as well as the submitted fresh (fixation in 10% buffered formalin, embedding in paraffin, preparation of 5 µm sections, staining with hematoxylin and eosin). Additionally, in dead birds parasitological, microbiological (blood, Gassner and Kimmig agar) and virological (3 passages in primary chicken embryo fibroblast and liver cell cultures as well as in SPF egg cultures, (Valo, Lohmann, Cuxhaven, Germany)) examinations were performed for the detection of pathogenic infectious agents by standard methods (Dufour-Zavala et al., 2008). The presence of Chlamydophila spp. were excluded by PCR [START_REF] Sachse | Detection and differentiation of Chlamydiae by nested PCR[END_REF]. All sera were tested for antibodies against paramyxovirus type 1 (PMV-1) by haemagglutination inhibition test (OIE, 2004).

Specific testing for avian bornavirus (ABV). Reverse transcriptase PCR (RT-PCR).

Organs and swabs were tested for the presence of ABV RNA by quantitative real-time RT-PCR (TaqMan) with two different primer sets [START_REF] Honkavuori | Novel Borna virus in psittacine birds with proventricular dilatation disease[END_REF]. In the case of negative results in these PCRs, but positive results in other tests a further primer set for standard PCR was used to detect other strains of ABV [START_REF] Enderlein | A strategy for reliable RT-PCR diagnosis of avian bornavirus infection[END_REF].

Immunohistochemistry (IHC).

The different organ samples of the dead psittacines were analysed immunohistochemically for the presence of ABV antigen by the avidin biotin complex (ABC) method using a polyclonal rabbit antibody directed against the phosphoprotein (p24) of the Borna disease virus (BDV) [START_REF] Herden | Comparison of different methods of diagnosing Borna disease in horses post mortem[END_REF][START_REF] Herzog | Indirect immunofluorescence assay suitable for intra vitam diagnosis of avian bornavirus infection in psittacine birds[END_REF].

Virus isolation. Organ samples were processed in infectivity assays according to the method described by Narayan et al. (1983). Briefly, tenfold dilutions of the organ homogenates (10%, Statistical analysis. The chi-square test was applied for analysis of differences between bird groups. With one degree of freedom, calculated values > 10.83 were considered to be highly significant (p<0.001) [START_REF] Dixon | Statistical software manual[END_REF].

Results

Post mortem diagnosis of PDD and ABV. In 37 out of 73 examined dead psittacines PDD was confirmed by histopathological detection of typical lesions of non-suppurative ganglioneuritis in various parts of the gastrointestinal tract often in combination with nonsuppurative encephalitis, neuritis, myelitis and myocarditis. The affected birds belonged to the bird genera Ara (n=10), Psittacus (7), Cacatua (6), Amazona (5), Eclectus (2), All these 37 cases were positive for ABV in at least one test (Table 3): In 26 cases, ABV RNA and antigen were detected by PCR and immunohistochemistry, respectively. The ABV antigen was detected in brain, spinal cord, retina, proventiculus, gizzard, and intestine by the anti-phophoprotein antibody. Additionally, ABV was isolated from brain, spinal cord and retina of 16 of these positive birds. Because of too small sample size or the condition of the tissues (beginning autolysis or formalin fixation/paraffin embedding), further 11 birds were only tested by RT-PCR or IHC, and were found to be positive for either ABV RNA (5) or antigen (6). Parasites, fungi, bacteria including chlamydia, and viruses other than ABV were not detected in any of the 37 birds.

The other 36 out of the 73 examined dead birds revealed no histopathological lesions typical for PDD (Table 3). In 29 of these birds neither ABV RNA nor antigen was detected by RT-PCR and immunohistological staining. Virus isolation attempts were performed in 15 of these cases with negative reults. However, seven (19%) of the birds without PDD lesions reacted positive for ABV in RT-PCR (4), IHC (1) or both tests (2).

The calculated value in chi-square test of 49.45 indicates highly significant differences between the groups and a highly significant association of PDD with the ABV infection (Table 3).

Intra vitam diagnosis of ABV.

From 1,442 live birds, 2,077 samples composed of crop and cloacal swabs and sera were received for direct and indirect detection of ABV. In RT-PCR, 86 of 347 crop swabs and 99 of 381 cloacal swabs were found to be positive for ABV RNA.

Of the 1349 sera tested, 228 revealed anti-ABV antibodies in IIFA, while all sera were negative for antibodies against PMV-1 in the HI test. Table 5 demonstrates the relation of anamnestic data with regard to PDD to ABV detection in the tested live birds. Birds with clinical signs of PDD (63) were positive for ABV to 66.6%. From 1379 birds without clinical signs of PDD, 81.4% were found to be negative for ABV, but in 257 birds (18.6%) ABV was detected directly or indirectly. The chi-quare value of 84.56 indicated that the differences between the groups were highly significant, and hence that ABV infection was associated with PDD at a high significance level.

Occurrence of ABV in countries and bird genera. ABV was detected in birds from all five

European countries from where birds or samples had been submitted for testing (Table 1).

The positive birds belonged to 33 of 54 different genera of the order Psittaciformes of which birds were examined (Table 2), and in 16 of them ABV infection was found for the first time.

In total 22.8% of all the psittacines reacted positive for ABV. Considering those genera with more than 30 birds tested, substancial differences in the percentages of ABV infected psittacines were seen. While the groups of Ara, Brotogeris and Eolophus contained about 20% positive reactors, the psittacines of the genera Aratinga (4.5%), Pyrrhura (9.0%), Psittacula (10%) and Agapornis (12.5%) revealed a notably lower infection rate. In contrast to this, higher percentages of ABV infected psittacines were observed in the genera Poicephalus (54.5%), Psittacus (46.5%), Cacatua (40.0%), Eclectus (36.2%) and Amazona (30.2%). The 12 non-psittacine birds included in this study were negative for ABV.

Discussion

In the present study, the results of testing a large number of different psittacine and some other bird species from various European countries on the occurrence of ABV infection were analysed and brought into relation with the available data on the PDD status in the tested birds. Additionally, the suitability of different specimens and test methods for ABV diagnostics were compared.

. In other studies for post mortem diagnosis of ABV infection [START_REF] Rinder | Broad tissue and cell tropism of avian bornavirus in parrots with proventricular dilatation disease[END_REF][START_REF] Gray | Use of avian bornavirus isolates to induce proventricular dilatation disease in conures[END_REF], Raghav et al., 2010), virus isolation, RT-PCR and/or IHC were performed only with freshly dead or euthanized birds or frozen organ samples. In this study, however, birds or samples of birds had been submitted for ABV diagnosis with different grades of freshness and different organ fixation. For this reason, organ samples of only 31 cases were suitable for virus isolation attempts without negative influence of toxic reactions and bacterial contamination in cell culture additionally to the use of RT-PCR and IHC. Organs of further 31 birds could be examined in RT-PCR and IHC, although autolysis in few cases hampered the IHC analysis due to high unspecific background staining. Tissue samples of 11 birds were already fixed in formalin or paraffin embedded and could be only processed immunohistochemically. These examinations indicated that RT-PCR and IHC are suitable test methods also for diagnostic material of different quality. To ensure optimal examination By intra vitam diagnostics for ABV about 20% of the nearly 1,500 birds were found to be positive for ABV RNA or displayed ABV-specific antibodies. The detection of more than 40% positive reactors in the group of birds with combined testing of three different samples, however, gave indication of a probably higher occurence of ABV infection in the population of captive psittacines. As in this group only one of five birds revealed to be positive in all three samples, testing of only one kind of sample may imply a high risk of false negative results. The observation that the presence of ABV RNA in cloacal excretions and anti-ABV antibodies in serum did not always coincide has already been made by [START_REF] Villanueva | The diagnosis of proventricular dilatation disease: Use of a Western blot assay to detect antibodies against avian Borna virus[END_REF] in a smaller number of birds. Possible explanations for seropositivity without detection of ABV RNA might be that i) the currently used RT-PCR are not able to detect all ABV genotypes [START_REF] Enderlein | A strategy for reliable RT-PCR diagnosis of avian bornavirus infection[END_REF] or that ii) the virus is only intermittently present in crop and/or cloaca which has been demonstrated for urofaeces of five ABV infected birds by daily testing over a period of 5 days (Raghav et al., 2010) or that iii) the virus was eliminated by the infected bird. ABV RNA detection in swabs of a bird without any humoral immune reaction might be due to sample taking at an early stage of infection or to an ABV infection which is able to hide from the immune system. Regarding these results, we strongly recommend the combined molecularbiological and serological testing of cloacal and crop swabs (eventually pooled) as well as serum for the diagnosis of ABV infection.

One further purpose of this study was to relate the results on the occurrence of ABV in dead and alive birds to their known status concerning signs of PDD. In post mortem examinations, other working groups [START_REF] Kistler | Recovery of divergent avian bornaviruses from cases of proventricular dilatation disease: identification of a candidate etiologic agent[END_REF][START_REF] Honkavuori | Novel Borna virus in psittacine birds with proventricular dilatation disease[END_REF][START_REF] Lierz | Anatomic distribution of avian Borna virus in parrots, its occurrence in clinically healthy birds and ABV antibody detection[END_REF]Ouyang et al., 2009;[START_REF] Rinder | Broad tissue and cell tropism of avian bornavirus in parrots with proventricular dilatation disease[END_REF], Weissenböck et al., 2009a;Raghav et al., 2010) However, we also found ABV RNA or antigen in psittacines without typical lesions of PDD.

Until now, this has only been observed in single birds by [START_REF] Lierz | Anatomic distribution of avian Borna virus in parrots, its occurrence in clinically healthy birds and ABV antibody detection[END_REF] and Raghav et al.

(2010) and may be an indication for inapparent infection with a low pathogenic ABV strain or an early stage of infection, when PDD lesions have not yet been induced. Nevertheless, these results strongly underline the causative role of ABV for the development of PDD, which has also been shown in first successfull experimental ABV infections [START_REF] Gancz | Experimental induction of proventricular dilatation disease in cockatiels (Nymphicus hollandicus) inoculated with brain homogenates containing avian bornavirus 4[END_REF][START_REF] Gray | Use of avian bornavirus isolates to induce proventricular dilatation disease in conures[END_REF].

By calculation in chi-square test, the relation of PDD to ABV infection was also clearly demonstrated as highly significant in live psittacines. The lower percentage (67%) of ABV positive birds wthin the group of PDD suspected birds in comparison to dead birds with confirmed PDD reflects the current difficulties in intra vitam diagnosis of PDD. The clinical signs of PDD like loss of weight, indigested grains in faeces, dilated proventriculus sometimes in combination with central nervous symptoms can also be seen in other diseases of the gastrointestinal tract and/or the nerval system [START_REF] Villanueva | The diagnosis of proventricular dilatation disease: Use of a Western blot assay to detect antibodies against avian Borna virus[END_REF][START_REF] Lierz | Die neuropathische Drüsenmagendilatation der Psittaziden und das aviäre Bornavirus als potentielle Ursache[END_REF]. Thus, it seems likely that not all of the birds in this study with clinically suspected PDD really suffer from this disease. However, because of the now proven association of ABV infection with PDD it is in all likelihood that a psittacine showing PDD like symptoms and being ABV positive is affected by PDD and not another disease.

Although the predominent part of the clinically healthy birds was negative for ABV, in nearly 20% of them ABV was detected directly and/or indirectly. These results further 2009), who tested a limited number of apparently healthy birds in a few aviaries, and found some of them to be ABV positve. Whether these birds were still in the incubation phase of the disease or remained only virus carriers without developing manifestation of clinical disease, as it is known for mammalian Borna disease [START_REF] Herden | Equine Borna disease[END_REF], can only be speculated at the present state of knowledge on ABV pathogenesis. It might be that similar immunopathological mechanisms like those which induce the outbreak of the Borna disease in mammals [START_REF] Richt | Borna disease[END_REF] may play an important role in the course of ABV infections. [START_REF] Rossi | Parrot proventricular dilatation disease[END_REF] suggest an autoimmune mechanism being involved in the development of clinical signs, as they found anti-ganglioside antibodies in PDD-affected birds and hypothesize that ABV might just serve as trigger for the development of the disease.

Concerning the occurrence of ABV in different countries ABV detections in captive psittacines have been reported from Israel, USA, Australia and Canada [START_REF] Kistler | Recovery of divergent avian bornaviruses from cases of proventricular dilatation disease: identification of a candidate etiologic agent[END_REF][START_REF] Honkavuori | Novel Borna virus in psittacine birds with proventricular dilatation disease[END_REF]Weissenböck et al., 2009;Raghav et al., 2010) as well as from various European countries like Germany [START_REF] Enderlein | Aviäres Bornavirus: Antikörper-und Genomnachweis in PDD positiven Vögeln[END_REF][START_REF] Lierz | Anatomic distribution of avian Borna virus in parrots, its occurrence in clinically healthy birds and ABV antibody detection[END_REF][START_REF] Rinder | Broad tissue and cell tropism of avian bornavirus in parrots with proventricular dilatation disease[END_REF], Austria, Switzerland and Hungary (Weissenböck et al., 2009a). In this study psittacines out of different bird collections in Spain, Italy, United Kingdom and Denmark were found to be positive for ABV, and the presence of ABV was further confirmed in German psittacines. Based on these findings it is likely that ABV is distributed worldwide in captive psittacine collections which may be due to the intense exchange and trade with these birds.

In total, about 20% of the 1,503 psittacines tested revealed signs of previous or ongoing ABV infection. These positive birds belong to 33 different psittacine genera (Table 2) and can be grouped as follows: i) 15 genera which are known to be affected by PDD, and ABV infection has been described in them before; they partly reveal remarkably high infection rates (e.g. Ara, Amazona, Poicephalus, Psittacus, Eclectus, Cacatua,) in the present Agapornis, Anodorhynchos, Psittacula); iii) one genus in which ABV has been reported before, but PDD is unknown so far, and iiii) further 12 genera in which neither PDD nor ABV infection has been reported previously (e.g. Bolborhynchus, Pyrrhura, Pionopsitta), and now were demonstrated to be ABV positve. Further systematic epidemiological investigations and experimental infection trials are necessary to investigate whether the observed differences are only due to the non representative mixture of psittacine birds examined or are caused by differences of psittacine genera in susceptibility for ABV infections.

  two independent research groups in Israel and USA identified a group of novel nonsegmented negative sense single-stranded RNA viruses by high throughput viral screens which were classified as four different genotypes of a new genus of the family Bornaviridae designated avian bornavirus (ABV)

  disease but it was not proven for any of these viral agents. However, results of two first experimental infections of a small number of cockatiels (Nymphicus hollandicus)

  shall provide further information on the suitability of different kinds of samples and the existing diagnostic tools for ABV detection as well as on the occurrence of ABV in different species and countries and its association with PDD.

  same range of organs) were processed for histopathological evaluation

  ) were prepared in GMEM medium (Gibco, Invitrogen, UK) plus 10% fetal bovine serum (FBS), mixed with equal volumes of freshly dispersed cells of the quail cell line CEC32 and incubated on chamber slides (Lab-Tek Products, Nunc, Roskilde, Denmark) for six days at 37°C. Virus replication was demonstrated by indirect immunofluorescence using polyclonal sera from experimentally BDV-infected rats cross-reacting with ABV[START_REF] Herzog | Indirect immunofluorescence assay suitable for intra vitam diagnosis of avian bornavirus infection in psittacine birds[END_REF].Immunofluorescence assay. The demonstration of anti-ABV serum antibodies (doubling dilutions of serum starting with a dilution of 1:10) were performed by an indirect immunofluorescence assay (IIFA) on persistently BDV-infected Madin-Darby canine kidney (MDCK) or ABV-infected CEC32 cells using a fluorescein isothiocyanate (FITC)-conjugated goat anti-bird IgG (Bethyl Laboratories, Montgomery, Texas) in a dilution of 1:50 according to[START_REF] Herzog | Indirect immunofluorescence assay suitable for intra vitam diagnosis of avian bornavirus infection in psittacine birds[END_REF].

  , Guaruba (1), Nandayus (1), Pionus (1), Barnardius (1), and Nymphicus (1).

  of samples were examined. In total 299 (20.7%) of the live birds reacted positive for ABV. The percentage of positive birds was even higher in those cases where several kinds of samples were investigated. As presented in Table4the ABV infection rate revealed to be markedly higher (121/276 birds = 43.8%), when crop and cloacal swab and serum from a single bird were tested in combination. While only about one fifth (25/121) of these birds showed positive reactions in all three samples submitted, the samples of the other cases were only partly positive for ABV: about one third (45) reacted only indirectly positive for ABV by exhibiting specific antibodies, another third (35) was only positive for ABV RNA in both swabs and 16/121 reactors proved to be positive in only one swab in combination with the serum or even alone.

  birds, which have to be send over long distances to the diagnostic laboratory it is proposed to transport the organ samples in an RNA stabilization reagent or in formalin fixed status.

  in a high percentage (up to 100%) of the PDD affected birds by different test methods, but without including non-affected birds. Due to the large number of birds with and without PDD in our examinations it was now possible for the first time by statistical analysis to prove this association as highly significant in naturally infected birds.

  support the findings of De Kloet & Dorresstein (2009),[START_REF] Lierz | Anatomic distribution of avian Borna virus in parrots, its occurrence in clinically healthy birds and ABV antibody detection[END_REF] andVillanueva et

  ) five genera which are known to be susceptible to PDD, and in which ABV now has been detected for the first time, some of them with only low percentages of positive birds (e.g.

  : immune-mediated viral ophthalmoencephalopathy causing blindness and behavioral abnormalities. Journal of Infectious Diseases, 148, 305-315. OIE (2004) Newcastle Disease. In OIE (Ed) Manual of Diagnostic Tests and Vaccines for Terrestrial Animals, 5 th edn (pp. 270-282), Paris, France. Orosz, S.E. & Dahlhausen, R.D (2007). Proventricular dilatation syndrome in an Amazon parrot: Possible role for PCR diagnostics of paramyxovirus-1 for presumptive diagnosis. Proceedings of the 9 th European AAV Conference-7 th Scientific Meeting (pp. 27-28). Zürich. Ouyang, N., Storts, R., Tian, Y., Wigle, W., Villanueva, I., Mirhosseini, N., Payne, S., Gray, P. & Tizard, I. (2009) Histopathology and the detection of avian bornavirus in the nervous system of birds diagnosed with proventricular dilatation disease. Avian Pathology, 38, 393-401. Phalen, D.N. (1986). An outbreak of psittacine proventricular dilatation syndrome (PPDS) in a private collection of birds and an atypical form of PPDS in a Nanday conure. Proceedings of the 7 th Annual Meeting of the Association of Avian Veterinarians (pp. 27-34). East Northport, N.Y., USA.Raghav, R., Taylor, M., DeLay, J., Ojkic, D., Pearl, D.L.,Kistler, A.L., DeRisi, J.L., Ganem, D., & Smith, D.A. (2010). Avian Bornavirus is present in many tisues of psittacine birds with histopathologic evidence of proventricular dilatation disease. Journal of Veterinary DiagnosticInvestigation, 22, 495-508. Reavill, D. & Schmidt, R.(2007). Lesions of the proventriculus/ventriculus of pet birds: 1640 cases. Proceedings of the 28 th Annual Meeting of the Association of Avian

F

F

F

  Table 4 demonstrates the results of testing for ABV RNA and/or antibodies with respect to the number of birds of which different

Table 1 .

 1 Origin and number of tested birds

	Country	Number of bird	Number of birds	Number of ABV
		collections		positive birds a
	Spain	14	996	142
	Germany	171	442	170
	Italy	12	32	17
	United Kingdom	16	16	5
	Denmark	2	29	9
	Total	215	1515	343
	a detection of avian bornavirus (ABV RNA, virus, antigen) and/or ABV specific antibodies

Table 2 .

 2 Bird genera examined for ABV infection

	Present study	Previous reports on

Table 3 .

 3 Association of PDD status and ABV detection in dead birds ABV positive: detection of ABV (RNA, virus, antigen) and/or ABV specific antibodies b PDD positive: pathohistological lesions typical for PDD * highly significant (p<0.001)

		ABV		Total number	% ABV
	PDD	positive a	negative	of birds	positive birds
	positive b	37	0	37	100
	negative	7	29	36	19
	Total	44	29	73
	Chi-square value: 49.45 *

a

Table 4 .

 4 Detection of ABV RNA and/or antibodies in swabs and sera of live birds

	Number of positive birds with

Table 5 .

 5 Association of PDD status and ABV detection in live birds

		ABV		Total number	% ABV
	PDD	positive a	negative	of birds	positive birds
	+ ? b	42	21	63	67
	-? c	257	1122	1379	19
	Total	299	1143	1442
	Chi-square value: 84.56 *

a ABV positive: detection of ABV (RNA, virus, antigen) and/or ABV specific antibodies b + ?: tentative diagnosis PDD: clinical signs like indigested feed, loss of weight, PDD in radiography, CNS disorder c -?: no report on clinical signs of PDD * highly significant (p<0.001)

E-mail: cavanagh@metronet.co.uk URL: http://mc.manuscriptcentral.com/cavp Avian Pathology

Acknowledgements

This research was supported by the Loro Parque Fundacion, Tenerife, Spain (Project No. PP-65-2009-1), and the Association for the Conservation of Threatened Parrots, Schöneiche, Germany. The authors are grateful to Jürgen Richt for kindly providing the antiphosphoprotein antibody .


Xavier De Ghellinck D'

Elseghem Vaernewijck

Keevin Didriche

Clement Lauzin

Atina Rizopoulos

Michel Herman
email: mherman@ulb.ac.be

Samir Kassi

Elseghem De Ghellinck D'

Vaernewijck

Prof M Herman

Cavity enhanced FTIR spectroscopy using a femto OPO absorption source

Keywords: Cavity enhanced absorption spectroscopy, FTIR, Femtosecond OPO comb spectroscopy, Femto-FT-CEAS Cavity enhanced absorption spectroscopy; FTIR, Femtosecond OPO comb spectroscopy, Femto-FT-CEAS

teaching and research institutions in France or abroad, or from public or private research centers.

Michelson-type Fourier transform interferometers (FTIR) are widely used in high resolution molecular infrared spectroscopy since pioneering studies such as those by, in alphabetical order, Brault (1,2), Delbouille (3,4), Guelachvili (5,6), Johns (7,8), Kauppinen (9,10), Valentin (11,12), Winnewisser [START_REF] Bp Winnewisser | Proc. SPIE-Int. Soc. Opt. Eng[END_REF]14) and their co-workers. More recently, new types of absorption schemes were developed to increase the signal and boost the detection sensitivity with FTIR instruments. For instance brighter absorption sources were used, such as synchrotron radiation (15) and broadband femto lasers (16,17), with conventional single or multipass absorption cells. Others were based on conventional sources with high finesse cavities to perform cavity enhanced absorption spectroscopy (CEAS) (18). Attempts to combine laser sources with CEAS and FTIR detection were also reported. Successful experiments include FT-CRDS (19,20) and FT-ICLAS (for intracavity laser absorption spectroscopy) (21,22) schemes. In 2010 cavity enhanced FTIR spectroscopy using a femtosecond laser absorption source was also reported (23,24), which we labeled Femto-FT-CEAS. The cavity modes comb was locked onto the Ti:Sa femto laser comb (the so-called "magic point (MP) (25,26)), detecting e.g. at ULB O 2 present in the atmosphere between the mirrors of the high finesse cavity open to air (23). We wish to report here on attempts to repeat such experiments, now using a tunable femto OPO absorption source and acetylene, ν 1 +ν 3 (the symmetric and antisymmetric CH stretching vibrations, respectively) as molecular band. In section 2, the successful use of a bright femto OPO absorption source for FTIR is presented, using various multipass arrangements. A more refined high-finesse cavity design is described in section 3 to achieve OPO-Femto-FT-CEAS.

Performances are further illustrated in the final section, which also concludes the present "proofs of principles"-type report. Although the present experiments will not involve locking the cavity comb onto the femto comb and are sometimes referred to as of integrated cavity output spectroscopy (ICOS) type, the latter is often associated to cavity off-axis experiments which is not the case here. We shall therefore keep using the more general CEAS terminology.

Use of a femto OPO absorption source

In our previous work,(23) a femto Ti:Sa source (Coherent Chameleon ULTRA II) served as absorption source. This laser is a mode locked laser with 80MHz repetition rate and 140 fs laser pulse width. We have now used this laser to pump an OPO module (Coherent PP810), allowing for broader wavelength coverage towards the IR. In principle, the full range 1 -3 µm is available if one also accounts for the idler option. It is, however limited by atmospheric water absorption which prevents laser operation in the lower IR We first checked that the use of the OPO module was compatible with the high resolution Bruker IFS120HR FTIR continuous acquisition procedure. The success of these experiments is illustrated in Figure 2 presenting a spectrum of acetylene in a supersonic expansion, recorded at 3 10

-2 cm -1
resolution (defined as 0.9/maximum optical path difference). A multipass optics made of two drilled gold mirrors was used, set to 25 passes in the expansion produced using a circular nozzle (0.5 mm inner diameter), the central hole in the mirrors allowing for light input and exit as previously described in the literature (27). We have used the latest version of the FANTASIO+ set-up (for "Fourier transform, tunable diode laser and mass spectrometers interfaced to a supersonic expansion") built at ULB to produce jet-cooled acetylene. This set-up is described in the literature and not further detailed (28,29). Precise experimental conditions are provided in the caption In a second step we achieved multipass absorption in a room temperature cavity. The cavity, 90 cm long, was identical to the one further described in the next section. The off-axis CEAS design was set to reach about 5 m absorption pathlength. The C 2 H 2 , ν 1 +ν 3 band was again recorded, now under room temperature conditions. A spectral portion is presented in (30). The regular series of lines missing in the simulation from 6535 to 6510 cm -1 is due to 13 CH 12 CH present in natural abundance in the sample (2%). The observation of this isotopologue is a nice illustration of the quality of the experimental data. The noise level reveals a minimal absorbance of 5 10 -3 , i.e. an absorption detection limit • min = 10 -5 cm -1 .

In a third step, we attempted to perform on-axis CEAS in a room temperature high finesse cavity. A major limitation was, however encountered, compared to our previous Ti:Sa experiments (23). It indeed turned out that the manufactured tuning mechanism involves a dispersion element that inherently adds chirp. This prevented us from efficiently locking together the cavity and OPO combs, thus from reaching the MP. This problem was not encountered in previous experiments using only the Ti:Sa pump source (23) since there is no similar dispersion element as in the OPO.

As a result all experiments were performed outside from the MP, by scanning the cavity length using a piezoelectric actuator (PZT) (HPST 1000 modulator) as described in the next section.

CEAS experiments

The set-up for CEAS experiments is schematized in Figure 4.

Broadband injection of a high-finesse cavity was achieved using a pair of lenses (L 1 and L 2 , f = 50 mm). The cavity was made of half inch output couplers (Layertec) stated to have 99.3 % reflectivity (CM 1 and CM 2 ). It corresponds to a finesse F =

). The output cavity mirror was set on a PZT. The cavity was about 77 cm long and the PZT was permanently modulating the cavity length. In this way, cavity modes are jittering and on the average every laser mode is equally transmitted, leading to a smooth transmission spectrum. Conditions need to be adapted empirically by tuning the cell length to a slightly different value for each selected central The laser beam was sent through a HoYag type optical Faraday isolator (Thorlabs) before injection in the cavity, to avoid optical feedback in the laser oscillator. This prooved to be essential in the success of the experiments.

The output laser beam was directed into the high resolution continuous scanning FTIR equipped with an InGaAs diode detector. The entrance FTIR iris aperture was set to 1.15 mm, i.e. larger than the laser beam diameter, the latter being focused to 10 µm at the FTIR entrance, using a 12.5 cm focal lens (L 3 ). Spectra are presented in Figures 5 and6, illustrating various features.

As shown in Figure 5, the modulation frequency of the PZT had to be set as high as possible (20 kHz) to reduce the noise level. This figure presents the same spectral range recorded using various PZT modulation frequencies.

The dominant contribution to the noise is actually likely to result from the beat between various frequencies with similar values, namely from the laser jitter, the PZT modulation and the FTIR sampling process. The resulting beat is smoothed out due to the detector response time (~ 1µs), actually lengthened by the use of a lowpass frequency filter. This effect is more efficient if the PZT The effect of the FTIR resolution is illustrated in Figure 6 with spectra recorded with 0.04, 0.02, 0.01 and 0.005 cm -1 resolution, from bottom to top. It is worth noting that a discretization of the spectrum starts appearing at 0.02 cm -1 FTIR resolution and is well present at 0.005 cm -1

FTIR resolution, giving rise to spike-like background signal. It is due to the free spectral range (FSR) of the fixed laser comb (FSR = 80 MHz), limiting the final resolution.

We also checked that S/N increases as the square root of the number of scans (n) as expected for white noise.

Data and conclusion

We used optimal conditions to record the ν 1 +ν 3 band in C 2 H 2 , as shown on Figure 7. It corresponds to an optimal rms noise equivalent absorption of 2.2 10 -9 cm -1 Hz -1/2 per spectral element, and a value • min of 1.5 10 These values are about 60 times better than those for the off-axis CEAS experiment previously reported (Figure 3). This improvement results from the increase in pathlength (30 times improvement) and also (another factor of 2) from the new design, involving an optical isolator and the fine tuning of the cavity, therefore decreasing the noise. This band and the many accompanying bands in the range have already been investigated in the literature using conventional FTIR multipass set-ups (e.g. (31)(32)(33)). Recently, ultra high sensitivity cavity ring down spectroscopy (CRDS) was applied in the range (30,34), revealing many more, weaker bands. The data recorded using Femto-FT-CEAS are of intermediate quality. We could spot lines not reported in the conventional FTIR investigations. Some of these were actually not assigned in the CRDS data, yet. Taking advantage on the very precise global modeling of the acetylene vibration-rotation energy levels in acetylene available from the literature and of the accurate resulting spectral simulations (30), we assigned low J-lines transitions in two bands, as reported in Table 1. All lines in these bands correspond to weak transitions, some overlapped by stronger lines and therefore missing in the Table. Some of the lines are highlighted in Figure 8.

In conclusion, the experiments just reported demonstrate that Femto-FT-CEAS can be successfully achieved using a tunable broadband OPO source. Various limitations existing with our present set-up were highlighted, Compared to CW-CRDS, sensitivity and resolution are not as high with Femto-FT-CEAS but recording speed and spectral bandwidth are significantly improved. Also, internal calibration procedure and data processing is ensured by the FTIR software and readily available. The many different possible recording conditions make a more quantitative comparison between CRDS and Femto-FT-CEAS difficult. It is clear, for instance that when combined to jet cooled experiments as used in our lab (28,29), both would address different types of problems, the former to be focused on exploring the absorption spectrum over a broad range, the latter to be dedicated to high sensitivity and high resolution investigations.

Two of the major advantages of Femto-FT-CEAS using a broadband OPO absorption source compared to more conventional FTIR approaches are that (i) thanks to the source brightness, high quality data can be recorded in a short amount of time, and (ii) the cell volume is much smaller (less than 200 cc in the present case) than for conventional multipass designs for identical or even larger pathlengths. We actually hope to benefit from the first of these by implementing Femto-FT-CEAS around the supersonic

  Figure merges the noise of all individual recordings. We shall here mainly

2 .

 2 We checked that the gain in S/N is about one order of magnitude compared to the use of a globar absorption source, other experimental conditions being identical.

Figure 3 (

 3 Figure 3 (top). Sharp negative signals corresponding to saturated peaks were

  directions for improvement. These include access to higher PZT modulation frequency and improvement of the mirrors quality (a reflectivity of 99.9%, in a larger bandwidth should be easily compatible with the actual set-up).

Figure 1 : 1 FWHM

 11 Figure 1: Illustration of the broad spectral coverage using a femto OPO

Figure 2 : 10 - 2 cm - 1 .

 21021 Figure 2:FTIR absorption spectrum of ν 1 +ν 3 , C 2 H 2 recorded using

Figure 3 :

 3 Figure 3: FTIR absorption spectrum of ν 1 +ν 3 , C 2 H 2 recorded using off-axis

Figure 4 :

 4 Figure 4: Experimental design for Femto-FT-CEAS using an OPO tunable

Figure 5 :

 5 Figure 5: FTIR absorption spectrum (arbitrary intensity units) of a femto

Figure 6 :

 6 Figure 6: Portion of the Femto-FT-CEAS spectrum of ν 1 +ν 3 , C 2 H 2 recorded

Figure 7 :

 7 Figure 7: FTIR spectrum of ν 1 +ν 3 , C 2 H 2 recorded using CEAS design and a

Figure 8 :

 8 Figure 8: Portions of the spectrum of ν 1 +ν 3 , C 2 H 2 shown on Figure 8. Newly

  The remaining noise at 20 kHz PZT modulation, which is channeling, can actually be removed from real spectra by producing a transmittance signal, i.e. dividing by I 0 . It is likely that even higher modulation will help in this problem. All other spectra presented here were recorded using 20 kHz modulation frequency.

	Molecular Physics	Page 8 of 29
	modulation is higher.
	F o
	r
	P
	e e r
	R e v i e w
	O n
	l y
	9

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Table 1 ,

 1 respectively.

	Page 20 of 29

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Table 1

 1

Table caption Table 1

 caption1 Transitions assigned in12 C 2 H 2 . The bands are identified using conventional numbering (30): v 1 v 2 v 3 v 4 v 5 , l 4 l 5 e/f, u/g. The predicted line wavenumbers

	Page 21 of 29			Molecular Physics
	Calc.	Ass.	Obs.	Obs.-Calc.
	(a)1 1 0 2 2, 2 0eg -0 0 0 1 1, 1 1eu
	6557.324 P5		6557.330	0.006
	6583.118 R5		6583.104	-0.014
	6589.857 R8		6589.849	-0.008
	6596.499 R11 6598.695 R12 (b) 1 1 0 2 2, 2 -2eg -0 0 0 1 1, 1 -1 6596.493 6598.685 6547.532 P10 6547.538 F o r	-0.006 -0.010 0.006
	6552.536 P8 6557.466 P6 6559.906 P5 6562.329 P4 6578.854 R2 6581.152 R3	6552.530 6557.460 6559.903 P 6562.327 6578.850 6581.153 e e r	-0.006 -0.006 -0.003 -0.001 -0.004 0.001
	6587.939 R6 6590.163 R7 6592.366 R8 6596.705 R10	6587.923 6590.163 6592.360 6596.691 R e -0.016 0.000 -0.006 -0.014 v i e w
					O n
					l y
	(from (30)), rotational assignments, observed and observed-predicted
	wavenumbers (all positions in cm -1 ) are provided.

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Acknowledgments

We are happy to dedicate this paper to Prof. Di Lonardo, for his many contributions to the acetylene spectroscopy, a dedication shared with all of us. We thank B. Amyay and P. Van Poucke (ULB) for their help on various items. We are indebted to the "programme Currien/Tournesol" collaboration sponsored by "Wallonie Bruxelles international », F.R.S.-FNRS and "Région Wallone", and 'Ministère des Affaires étrangères" and "Ministère de l'Education Nationale, de la Recherche et de la Technologie" on the Belgian and French sides, respectively. This work was also sponsored in Belgium, by the Fonds National de la Recherche Scientifique (FNRS, contracts FRFC and IISN) and the « Action de Recherches Concertées de la Communauté française de Belgique ».