Experimental Infection of Embryonic Cells and Embryonated Eggs of Cockatiels (Nymphicus hollandicus) with Two Parrot Bornavirus Isolates (PaBV-4 and PaBV-2).

Parrot bornavirus (PaBV) might be transmitted vertically. Cockatiel embryonic brain cells and embryonated eggs of cockatiels (ECE) were infected with PaBV-2 and PaBV-4. In embryonic brain cells, PaBV-2 and PaBV-4 showed no differences in viral spread despite the slower growth of PaBV-2 compared with PaBV-4 in CEC-32 cells. ECE were inoculated with PaBV-4 and 13-14 dpi, organs were sampled for RT-PCR, immunohistochemistry/histology, and virus isolation. In 28.1% of the embryos PaBV-4-RNA and in 81.3% PaBV-4-antigen was detected in the brain. Virus isolation failed. Division of organ samples and uneven tissue distribution of the virus limited the results. Therefore, 25 ECE were inoculated with PaBV-4 (group 1) and 15 ECE with PaBV-2 (group 3) in the yolk sac, and 25 ECE were inoculated with PaBV-4 (group 2) and 15 eggs with PaBV-2 (group 4) in the chorioallantoic membrane to use the complete organs from each embryo for each examination method. PaBV-RNA was detected in the brain of 80% of the embryos in groups 1, 2, 3 and in 100% of the embryos in group 4. In 90% of the infected embryos of group 1, and 100% of group 2, 3 and 4, PaBV antigen was detected in the brain. PaBV antigen-positive brain cells were negative for anti-neuronal nuclear protein, anti-glial fibrillary acidic protein, and anti S-100 staining. Virus was not re-isolated. These results demonstrated a specific distribution pattern and spread of PaBV-4 and PaBV-2 in the brain when inoculated in ECE. These findings support a potential for vertical transmission.

A Novel Dipotassium Hydrogen Phosphate Phantom for Calibrating Computed Tomographic Bone Density Measurements in Birds.

The objective of this study was to construct a calibration phantom for bone mineral density (BMD) measurements adapted to avian anatomy by quantitative computed tomography. The determination of BMD is important to assess avian osteoporosis in poultry at production facilities and to study biological features in association with flight patterns in birds. Quantitative computed tomography measured in Hounsfield units is a well-established technique for BMD measurements. Translation of Hounsfield units into the International System of Units (mg/cm3) requires the use of a calibration phantom. Although calibration phantoms for routine use in humans are commercially available, phantoms suited to avian anatomy are not. A liquid dipotassium hydrogen phosphate calibration standard was constructed out of commercially available materials, easily allowing for variations in size, bone diameter, and adaptation to avian skeletal anatomy. Periodically, quantitative computed tomography scans were performed to monitor constant correlation to the calibration standard over 3 months and to monitor for the potential influence of gas bubbling and water evaporation in the rods on BMD measurements. Finally, the calibration phantom was tested for BMD measurements with carcasses from 2 bird species, including 3 peregrine falcons (Falco peregrinus; 2 juvenile males, 1 adult female with inactive reproductive status) and 4 Eurasian kestrels (Falco tinnunculus; 1 juvenile and 2 adult males, 1 adult female with inactive reproductive status). Results demonstrated stability of the calibration phantom without the need to refill or replace rods, plus a stable correlation line (R 2 = .99) over the 3-month evaluation period. It was possible to place the phantom directly on the bird carcasses, close to the measured bones, to improve BMD analysis. As evaluated, the phantom appeared to be adaptive to avian skeletal anatomy. Moreover, it was possible to build the phantom within 24 hours from commercially available materials.

Age-dependent development and clinical characteristics of an experimental parrot bornavirus-4 (PaBV-4) infection in cockatiels (Nymphicus hollandicus).

Parrot bornavirus (PaBV) is a pathogen often found in psittacine populations. Infected, clinically healthy carrier birds are of major importance for epidemiology, but the underlying pathomechanism of this carrier status is poorly understood. The age, implying the maturation status of the immune system, at the time of infection might be significant for the clinical outcome. Therefore, two groups of 11 cockatiels of different ages (adult and newly hatched) were inoculated with a PaBV-4 isolate intravenously. The trial lasted for 233 days and all birds were observed for clinical signs, PaBV-RNA shedding and anti-PaBV antibody production. At the end of the trial, histopathology, immunohistochemistry, PCR and virus re-isolation were performed. All 22 birds seroconverted and shed PaBV-RNA during the investigation period; the juvenile group earlier and more homogeneously. Nine of 11 birds of the adult group developed clinical signs; five birds died or had to be euthanized before the end of the study. In the juvenile group none of the birds developed clinical signs and only one bird died due to bacterial septicaemia. Eight birds of the adult group, but none of the juvenile group, showed a dilatation of the proventriculus. PaBV-RNA detection and virus re-isolation were successful in all birds. Immunohistochemically, PaBV antigen was found in all birds. Histopathology revealed mononuclear infiltrations in organs in birds of both groups, but the juveniles were less severely affected in the brain.Thus, PaBV infection at an age with a more naïve immune system makes the production of carrier birds more likely.RESEARCH HIGHLIGHTS PaBV infection at a young age might favour the development of carrier birds.Cockatiels infected at a very young age showed inflammation but no clinical signs.The juvenile group started seroconversion and PaBV-RNA shedding earlier.Seroconversion and PaBV-RNA shedding occurred more homogeneously in the juveniles.

Wounds as the Portal of Entrance for Parrot Bornavirus 4 (PaBV-4) and Retrograde Axonal Transport in Experimentally Infected Cockatiels (Nymphicus hollandicus).

In this study, we investigated the natural route of infection of psittacine bornavirus (PaBV), which is the causative agent of proventricular dilatation disease (PDD) in psittacines. We inoculated two infection groups through wounds with a PaBV-4 isolate. In nine cockatiels (Nymphicus hollandicus) we applied a virus suspension with a titer of 103 50% tissue culture infection dose (TCID50) via palatal lesions (Group P, P1-9). In a second group of three cockatiels, we applied a virus suspension with a titer of 104 TCID50 to footpad lesions (Group F, F1-3). In two cockatiels, the control (or "mock") group, we applied a virus-free cell suspension (Group M, M1-2) via palatal lesions. The observation period was 6 mo (Groups P and M) or 7 mo (Group F). We monitored PaBV-4 RNA shedding and seroconversion. At the end of the study, we examined the birds for the presence of inflammatory lesions, PaBV-4 RNA, and antigen in tissues, as well as virus reisolation of brain and crop material. We did not observe any clinical signs typical of PDD during this study. We also did not see seroconversion or PaBV RNA shedding in any bird during the entire investigation period, and virus reisolation was not successful. We only found PaBV-4 RNA in sciatic nerves, footpad tissue, skin, and in one sample from the intestine of Group F. In this group, the histopathology revealed mononuclear infiltrations mainly in skin and footpad tissue; immunohistochemistry showed positive reactions in spinal ganglia and in the spinal cord, and slightly in skin, footpad tissues, and sciatic nerves. In Groups P and M we found no viral antigen or specific inflammations. In summary, only the virus application on the footpad lesion led to detectable PaBV RNA, mononuclear infiltrations, and positive immunohistochemical reactions in tissues of the experimental birds. This could suggest that PaBV spreads via nervous tissue, with skin wounds as the primary entry route.

Description, occurrence and significance of Mycoplasma seminis sp. nov. isolated from semen of a gyrfalcon (Falco rusticolus).

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

Correlation of avian bornavirus-specific antibodies and viral ribonucleic acid shedding with neurological signs and feather-damaging behaviour in psittacine birds.

Parrot bornaviruses (PaBV) are the causative agents of proventricular dilatation disease in psittacine birds, but have also been linked to other clinical signs, including behavioural disorders and neurological signs. The aim of this study was to correlate PaBV infection in birds showing feather-damaging behaviour or neurological signs for which no other cause of disease could be identified. Psittacine birds presented to a private practice were divided into three groups: birds with neurological signs (n=28), birds showing feather-damaging behaviour (n=42) and birds presented for routine examinations (n=56). Swabs of crop and cloaca were collected and investigated for the presence of PaBV-RNA using real time RT-PCR. Additionally, serum samples were taken and examined for the presence of anti-PaBV antibodies by immunofluorescence test. PaBV infection was detected in one of the test systems in 40.5 per cent of all birds (n=126) investigated. In the clinically healthy birds (n=56), 19.6 per cent of the birds were positive in at least one of the PaBV tests, compared with 52.38 per cent of the feather-damaging (n=42) and 64.28 per cent of the neurologically diseased birds (n=28). Interestingly, the anti-PaBV antibody titres in birds with neurological signs were highest up to 1:20 480. High antibody titres (up to 1:5120) were also found in the feather-damaging group, whereas the birds of the control group, if PaBV positive, had only very low titres. Similarly, the highest viral load was found in the group of the neurologically diseased birds, followed by feather-damaging birds, whereas PaBV-positive birds in the control group demonstrated only low viral RNA shedding. A clear correlation between severity of clinical signs, amount of viral shedding and high levels of antibody titres was observed for most of the neurologically diseased birds and also for few birds with feather-damaging behaviour. For the first time, these results clearly indicate a correlation between PaBV infection and neurological signs in birds without gastrointestinal signs presented to the veterinarian in practice. It also may demonstrate a possible correlation with feather-damaging behaviour and anti-PaBV antibody presence. The antibody titre seems to represent a diagnostic tool to correlate clinical signs to PaBV as a cause.

Prevalence of Sarcocystis calchasi in free-ranging host species: Accipiter hawks and Common Woodpigeon in Germany.

The apicomplexan parasite Sarcocystis calchasi (S. calchasi) triggers pigeon protozoal encephalitis, a neurologic disease in columbids. Accipiter hawks have been identified as the final host, and Columbidae and Psittaciformes as intermediate hosts. In this study, 368 free-ranging Accipiter hawks and 647 free-ranging common woodpigeons were sampled in a country-wide study in order to identify the prevalence of S. calchasi in these populations. A semi-nested PCR specific for S. calchasi tested positive in 7.3% (4.9-10.5) of submitted samples from Accipiter hawks. Juvenile Accipiter hawks (13.7%; 7.7-22.0) had a significantly higher infection rate with S. calchasi than adult Accipiter hawks (5.8%; 2.7-9.3). The prevalence of S. calchasi in common woodpigeons was 3.3% (5.4-9.7). Positive pigeons were identified in 14/16 federal states, and a region-dependency was detected, with higher rates of infection in the eastern parts of Germany. The results of this study suggest that the common woodpigeon is a natural reservoir for S. calchasi. In a study of one region for four consecutive years, an increase in prevalence was not detected. Findings indicate that the parasite is not newly introduced to Germany, but rather long established. The prevalence suggests that there is a substantial risk of S. calchasi infections in other free-ranging as well as captive host species.

Investigation of Different Infection Routes of Parrot Bornavirus in Cockatiels.

The aim of this study was to determine the natural infection route of parrot bornavirus (PaBV), the causative agent of proventricular dilatation disease (PDD) in psittacines. For this purpose, nine cockatiels ( Nymphicus hollandicus ) were inoculated orally, and nine cockatiels were inoculated intranasally, with a PaBV-4 isolate. To compare the results of the trials, the same isolate and the same experimental design were used as in a previous study where infection was successful by intravenous as well as intracerebral inoculation. After inoculation, the birds were observed for a period of 6 mo and tested for PaBV RNA shedding, virus replication, presence of inflammatory lesions, and PaBV-4 antigen in tissues, as well as specific antibody production. In contrast to the previous study involving intravenous and intracerebral infections, clinical signs typical for PDD were not observed in this study. Additionally, anti-PaBV antibodies and infectious virus were not detected in any investigated bird during the study. Parrot bornavirus RNA was detected in only four birds early after infection (1-34 days postinfection). Furthermore, histopathologic examination did not reveal lesions typical for PDD, and PaBV antigen was not detected in any organ investigated by immunohistochemistry. In summary, oral or nasal inoculation did not lead to a valid infection with PaBV in these cockatiels. Therefore it seems to be questionable that the formerly proposed fecal-oral transmission is the natural route of infection in immunocompetent adult or subadult cockatiels.

Description and prevalence of Mycoplasma ciconiae sp. nov. isolated from white stork nestlings (Ciconia ciconia).

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

Accipiter hawks (Accipitridae) confirmed as definitive hosts of Sarcocystis turdusi, Sarcocystis cornixi and Sarcocystis sp. ex Phalacrocorax carbo.

Sarcocystis is a large genus of protozoan parasites with complex heteroxenous life cycles. For many species, either the intermediate or the definitive host is still unknown. In this study, 116 Accipiter hawks (Eurasian sparrowhawks and northern goshawks) were investigated for the presence of Sarcocystis spp. in their intestinal tract or their faeces. To gain a wide distribution, samples were collected throughout Germany within 2 years. It was possible to detect Sarcocystis-like oocysts in 65 samples. Sequencing of the ITS region or species-specific PCR identified 33 samples as Sarcocystis turdusi/Sarcocystis sp. ex A. nisus (18), Sarcocystis calchasi (6), Sarcocystis columbae (3), Sarcocystis cornixi (3) and Sarcocystis sp. ex Phalacrocorax carbo (3). Besides the known infestation with S. columbae, S. sp. ex A. nisus and S. calchasi the Accipiter hawks were thereby confirmed as definitive host of S. turdusi, S. cornixi and S. sp. ex Phalacrocorax carbo for the first time.

Parrot Bornavirus (PaBV)-2 isolate causes different disease patterns in cockatiels than PaBV-4.

Psittaciform 1 bornavirus (PaBV) has already been shown to be the aetiologic agent of proventricular dilatation disease, a significant disease of birds. However, the pathogenesis of PaBV infection has not yet been resolved and valid data regarding the pathogenicity of different PaBV species are lacking. Thus, the present study was aimed to characterize the influence of two different PaBV species on the course of disease. Eighteen cockatiels were inoculated intracerebrally (i.c.) or intravenously (i.v.) with a PaBV-2 isolate under the same conditions as in a previous study using PaBV-4. Birds were surveyed and sampled for 33 weeks to analyse the course of infection and disease in comparison to that of PaBV-4. Similar to PaBV-4, PaBV-2 induced a persistent infection with seroconversion (from day 6 p.i. onwards) and shedding of viral RNA (from day 27 p.i. onwards). However, in contrast to PaBV-4, more birds displayed clinical signs and disease progression was more severe. After PaBV-2 infection, 12 birds exhibited clinical signs and 10 birds revealed a dilated proventriculus in necropsy. After PaBV-4 infection only four birds revealed clinical signs and seven birds showed a dilatation of the proventriculus. Clinically, different courses of disease were observed after PaBV-2 infection, mainly affecting the gastrointestinal tract. This had not been detected after PaBV-4 infection where more neurological signs were noted. The results provide evidence for different disease patterns according to different PaBV species, allowing the comparison between the infection with two PaBV species, and thus underlining the role of viral and individual host factors for disease outcome.

Parasite distribution and early-stage encephalitis in Sarcocystis calchasi infections in domestic pigeons (Columba livia f. domestica).

Pigeon protozoal encephalitis is a biphasic, neurologic disease of domestic pigeons (Columba livia f. domestica) caused by the apicomplexan parasite Sarcocystis calchasi. Despite severe inflammatory lesions of the brain, associated parasitic stages have only rarely been identified and the cause of the lesions is still unclear. The aim of this study was therefore to characterize the tissue distribution of S. calchasi within pigeons between the two clinical phases and during the occurrence of neurological signs. For this purpose, a semi-quantitative real-time polymerase chain reaction (PCR) was developed. Forty-five domestic pigeons were infected orally (via a cannula into the crop) with 200 S. calchasi sporocysts and euthanized in groups of three pigeons at intervals of 2 to 10 days over a period of 61 days. Tissue samples including brain and skeletal muscle were examined by histology, immunohistochemistry, and PCR. Schizonts were detected in the liver of one pigeon at day 10 post infection. A mild encephalitis was detected at day 20 post infection, around 4 weeks before the onset of neurological signs. At the same time, immature sarcocysts were present in the skeletal muscle. In seven pigeons a few sarcocysts were identified in the brain, but not associated with any lesion. These results suggest that the encephalitis is induced at a very early stage of the S. calchasi lifecycle rather than in the chronic phase of pigeon protozoal encephalitis. Despite the increasing severity of lesions in the central nervous system, the amount of sarcocysts did not increase. This supports the hypothesis of a delayed-type hypersensitivity response as the cause of the encephalitis. The study also demonstrated that S. calchasi DNA is detectable in tissues negative by histological methods, indicating a higher sensitivity of the real-time PCR.

Avian bornavirus in free-ranging psittacine birds, Brazil.

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

Follow-up investigations on different courses of natural avian bornavirus infections in psittacines.

To study the course of natural avian bornavirus (ABV) infection, 63 psittacines of three bird collections where ABV had been demonstrated were investigated over a period of 1 yr. The psittacines were clinically observed and swabs of crop and cloaca as well as serum samples were collected three separate times at intervals of 2-6 mo. According to the results of detection of ABV RNA by reverse transcriptase polymerase chain reaction (RT-PCR) and of anti-ABV antibodies by indirect immunofluorescence assay (IIFA), 43 of the birds were found to be infected with ABV. Based on variations in virus shedding and antibody production in combination with the occurrence of proventricular dilatation disease (PDD) -related clinical signs, pathological findings, and lethal outcome, four different groups of infected psittacines and a fifth group of noninfected psittacines were identified. Group 1 comprised six birds with various courses of ABV infection and forms of clinical PDD. Groups 2-4 included all birds with subclinical ABV infections: Group 2 contained 13 birds that were consistently (subgroup A, 6 birds) or inconsistently (subgroup B, 7 birds) ABV positive by PCR and serology; group 3 was composed of 13 psittacines exhibiting only anti-ABV antibodies; and 8 birds that had positive ABV RNA detection in crop and cloaca, but did not develop anti-ABV specific antibodies, were classified in group 4. Twenty-three out of the 63 psittacines remained free of detectable ABV RNA or anti-ABV antibodies over the whole observation period (group 5). Based on the results, it seems that birds with high ABV RNA load in crop and cloaca combined with high anti-ABV antibodies have a high risk of the development of PDD, indicating that the humoral antibodies do not protect against the disease. The meaning of the detection of ABV RNA and antibodies at a low and inconsistent level for the single bird as well as for the epidemiology of the ABV infection remained unclear in this field study and needs to be further investigated.

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.

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.

The structure of avian polyomavirus reveals variably sized capsids, non-conserved inter-capsomere interactions, and a possible location of the minor capsid protein VP4.

Avian polyomavirus (APV) causes a fatal, multi-organ disease among several bird species. Using cryogenic electron microscopy and other biochemical techniques, we investigated the structure of APV and compared it to that of mammalian polyomaviruses, particularly JC polyomavirus and simian virus 40. The structure of the pentameric major capsid protein (VP1) is mostly conserved; however, APV VP1 has a unique, truncated C-terminus that eliminates an intercapsomere-connecting ß-hairpin observed in other polyomaviruses. We postulate that the terminal ß-hairpin locks other polyomavirus capsids in a stable conformation and that absence of the hairpin leads to the observed capsid size variation in APV. Plug-like density features were observed at the base of the VP1 pentamers, consistent with the known location of minor capsid proteins VP2 and VP3. However, the plug density is more prominent in APV and may include VP4, a minor capsid protein unique to bird polyomaviruses.

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.