Dominik Fischer

Pathogenesis of West Nile virus lineage 1 and 2 in experimentally infected large falcons.

West Nile virus (WNV) is a zoonotic flavivirus that is transmitted by blood-suckling mosquitoes with birds serving as the primary vertebrate reservoir hosts (enzootic cycle). Some bird species like ravens, raptors and jays are highly susceptible and develop deadly encephalitis while others are infected subclinically only. Birds of prey are highly susceptible and show substantial mortality rates following infection. To investigate the WNV pathogenesis in falcons we inoculated twelve large falcons, 6 birds per group, subcutaneously with viruses belonging to two different lineages (lineage 1 strain NY 99 and lineage 2 strain Austria). Three different infection doses were utilized: low (approx. 500 TCID50), intermediate (approx. 4 log10 TCID50) and high (approx. 6 log10 TCID50). Clinical signs were monitored during the course of the experiments lasting 14 and 21 days. All falcons developed viremia for two weeks and shed virus for almost the same period of time. Using quantitative real-time RT-PCR WNV was detected in blood, in cloacal and oropharyngeal swabs and following euthanasia and necropsy of the animals in a variety of neuronal and extraneuronal organs. Antibodies to WNV were first time detected by ELISA and neutralization assay after 6 days post infection (dpi). Pathological findings consistently included splenomegaly, non-suppurative myocarditis, meningoencephalitis and vasculitis. By immunohistochemistry WNV-antigens were demonstrated intralesionally. These results impressively illustrate the devastating and possibly deadly effects of WNV infection in falcons, independent of the genetic lineage and dose of the challenge virus used. Due to the relatively high virus load and long duration of viremia falcons may also be considered competent WNV amplifying hosts, and thus may play a role in the transmission cycle of this zoonotic virus.

Epidemic Spread of Usutu Virus in Southwest Germany in 2011 to 2013 and Monitoring of Wild Birds for Usutu and West Nile Viruses

Mosquito-borne viruses are becoming an increasing threat for Europe. One of these viruses is Usutu virus (USUV), a single-stranded RNA virus belonging to the Japanese encephalitis virus group within the family Flaviviridae. Since the occurrence of USUV among wild birds in June, 2011, infected Blackbirds (Turdus merula) have frequently been found dead in southwest Germany, cumulating in a massive die-off. Moreover, other bird species (Strigiformes) in this region have been affected. In a first study, 209 of over 600 dead birds (wild birds and birds kept in aviaries) collected from 2011 to 2013 carried USUV, more than 88% of them Blackbirds. USUV had already been detected in 2010, one year before the epizooty, in a mosquito-based surveillance program in Germany. The main epidemic area of the USUV outbreak in wild birds in southwest Germany has been similar for the last three years. In a second study during 2011 to 2013, 902 live migratory and resident birds (representing 87 bird species belonging to 14 bird orders) from four different sampling sites were bled and tested serologically and by qPCR for West Nile virus (WNV) and USUV infections. No USUV or WNV genomes were detected. Some migratory birds (mainly long-distance migrants and some partial migrants) carried neutralizing antibodies against WNV as discriminated by USUV and WNV cross-neutralization tests. Only few resident birds showed relevant USUV-specific neutralizing antibodies. The occurrence of USUV in the Upper Rhine valley area of southwest Germany is a proof of principle for the incursion and spread of other arthropod-borne (arbo)-viruses along these routes. Therefore, monitoring studies in birds and mosquitoes for the presence of arboviruses in these areas are indispensable.

Diving-Flight Aerodynamics of a Peregrine Falcon (Falco peregrinus)

This study investigates the aerodynamics of the falcon Falco peregrinus while diving. During a dive peregrines can reach velocities of more than 320 km h−1. Unfortunately, in freely roaming falcons, these high velocities prohibit a precise determination of flight parameters such as velocity and acceleration as well as body shape and wing contour. Therefore, individual F. peregrinus were trained to dive in front of a vertical dam with a height of 60 m. The presence of a well-defined background allowed us to reconstruct the flight path and the body shape of the falcon during certain flight phases. Flight trajectories were obtained with a stereo high-speed camera system. In addition, body images of the falcon were taken from two perspectives with a high-resolution digital camera. The dam allowed us to match the high-resolution images obtained from the digital camera with the corresponding images taken with the high-speed cameras. Using these data we built a life-size model of F. peregrinus and used it to measure the drag and lift forces in a wind-tunnel. We compared these forces acting on the model with the data obtained from the 3-D flight path trajectory of the diving F. peregrinus. Visualizations of the flow in the wind-tunnel uncovered details of the flow structure around the falcon’s body, which suggests local regions with separation of flow. High-resolution pictures of the diving peregrine indicate that feathers pop-up in the equivalent regions, where flow separation in the model falcon occurred.

Comparison of Diagnostic Tools for the Detection of Aspergillosis in Blood Samples of Experimentally Infected Falcons

SUMMARY Antemortem diagnosis of avian aspergillosis is very challenging. Diagnostic assays using blood samples would aid in an early and more definitive diagnosis. In the current study, detection of anti-Aspergillus antibodies, Aspergillus antigen, and Aspergillus toxin (fumigaclavine A), protein electrophoresis and measurement of acute-phase protein concentrations were performed on serum of 18 adult and plasma of 21 juvenile gyr–saker hybrid falcons (Falco rusticolus × Falco cherrug). Adult (n  =  15) and juvenile (n  =  18) falcons were experimentally inoculated with different dosages of the same strain of Aspergillus fumigatus and an additional three falcons from each age group were used as uninfected control animals. Blood samples were collected prior to inoculation and at 28 days postinoculation. Of the 33 inoculated falcons, 16 demonstrated clinical signs (vomiting, greenish urates, dyspnea, ruffled feathers) commonly associated with aspergillosis and in 14 falcons necropsy revealed aspergillosis granulomas confirmed by mycology and histopathology. Positive galactomannan results were rare, with only 3/15 positive samples from adult falcons and none in the juvenile birds. Most of the inoculated falcons showed an increase of serum amyloid A (66.7%) and haptoglobin (70.4%), but fumigaclavine A was not detected in the blood from any of the experimental animals. Elevated antibody indices were detected in 96.7% of the inoculated birds, but also in 66.7% of the controls. Significant decreases in albumin∶globulin ratio were obvious in 81.5% of the inoculated birds, including 100% of the birds with granulomas. Blood from falcons with granulomas demonstrated significantly increased concentration values of alpha 2 and β globulins, decreased percentages of prealbumin and albumin, and increased percentages of alpha 2 and β globulins compared to inoculated falcons without granulomas. In conclusion, acute-phase proteins and the electrophoretic profile of birds challenged with A. fumigatus show significant alterations, which in combination with other diagnostic procedures, assist in the early diagnosis of avian aspergillosis. RESUMEN Comparación de las herramientas de diagnóstico para la detección de la aspergilosis en muestras de sangre de halcones infectados experimentalmente. El diagnóstico antemortem de la aspergilosis aviar es muy difícil. Los ensayos de diagnóstico utilizando muestras de sangre podrían ayudar en el diagnóstico precoz y definitivo. En el estudio actual, se llevó a cabo la detección de anticuerpos anti-Aspergillus, la detección del antígeno de Aspergillus, y de la toxina de Aspergillus (fumigaclavina A), el análisis de proteínas por electroforesis y la medición de las concentraciones de proteínas de la fase aguda en suero de 18 halcones adultos y en 21 muestras de plasma de halcones juveniles híbridos gyr-saker (Falco rusticolus × Falco cherrug). Halcones adultos (n  =  15) y juveniles (n  =  18) fueron inoculados experimentalmente con diferentes dosis de la misma cepa de Aspergillus fumigatus y otros tres halcones adicionales de cada grupo de edad fueron utilizados como animales control no infectados. Se recolectaron muestras de sangre antes de la inoculación y a los 28 días después de la inoculación. De los 33 halcones inoculados, 16 mostraron signos clínicos (vómitos, excreción de uratos verdosos, disnea, plumas erizadas) comúnmente asociados con la aspergilosis y la necropsia de 14 halcones reveló granulomas por aspergilosis confirmados por el estudio de micología e histopatología. Resultados positivos de galactomananos fueron raros, con sólo tres de quince muestras positivas de los halcones adultos y ninguna de las aves jóvenes. La mayoría de los halcones inoculados mostraron un aumento del amiloide A sérico (66.7%) y de la haptoglobina (70.4%), pero no se detectó la fumigaclavina A en la sangre de ninguno de los animales de experimentación. Se detectaron índices elevados de anticuerpos en 96.7% en las aves inoculadas, pero también en el 66.7% de los controles. Fueron evidentes las disminuciones significativas en la relación albúmina∶globulina en el 81.5% de las aves inoculadas, incluyendo al 100% de las aves con granulomas. La sangre de los halcones con granulomas mostró un aumento significativo de la concentración de alfa-2 y β globulinas, una disminución de los porcentajes de prealbúmina y albúmina, y un aumento de los porcentajes de alfa 2 y β globulinas en comparación con los halcones inoculados sin granulomas. En conclusión, las proteínas de fase aguda y el perfil electroforético de las aves desafiadas con A. fumigatus muestran alteraciones significativas, que en combinación con otros procedimientos de diagnóstico, pueden ayudar en el diagnóstico precoz de la aspergilosis aviar.

Susceptibility of adult pigeons and hybrid falcons to experimental aspergillosis.

Aspergillosis caused by Aspergillus fumigatus seems to be more prevalent in some avian species than in others. We compared the development of aspergillosis in 8-month-old Gyr-Saker hybrid falcons and 8-month-old pigeons after a single intratracheal inoculation of different dosages of A. fumigatus conidia (107, 105 and 103). Clinical signs, including vomiting, discoloration of the urates, loss of appetite and dyspnoea, were observed in four out of five falcons and in four out of five pigeons inoculated with 107 A. fumigatus conidia. Necropsy revealed the presence of granulomas in the air sacs and/or lungs in four out of five falcons and in four out of five pigeons in the high dosage group. A. fumigatus was isolated from these granulomas in three falcons and in three pigeons. The presence of fungal hyphae was detected with Periodic acid Shiff reagent staining in three out of five falcons and in three out of five pigeons in the high dosage group. Avian respiratory macrophages were clearly present in and around the fungal granulomas. In the other dosage groups, no granulomas, positive A. fumigatus cultures or fungal hyphae were present, except for one falcon in the middle dosage group in which a sterile granuloma without fungal hyphae was noticed. In conclusion, the study shows that adult falcons and pigeons are susceptible to aspergillosis after inoculation of a single dose of conidia intratracheally.

Limited efficacy of West Nile virus vaccines in large falcons (Falco spp.)

West Nile virus (WNV) can lead to fatal diseases in raptor species. Unfortunately, there is no vaccine which has been designed specifically for use in breeding stocks of falcons. Therefore the immunogenicity and protective capacity of two commercially available WNV vaccines, both approved for use in horses, were evaluated in large falcons. One vaccine contained adjuvanted inactivated WNV lineage 1 immunogens, while the second represented a canarypox recombinant live virus vector vaccine. The efficacy of different vaccination regimes for these two vaccines was assessed serologically and by challenging the falcons with a WNV strain of homologous lineage 1. Our studies show that the recombinant vaccine conveys a slightly better protection than the inactivated vaccine, but moderate (recombinant vaccine) or weak (inactivated vaccine) side effects were observed at the injection sites. Using the recommended 2-dose regimen, both vaccines elicited only sub-optimal antibody responses and gave only partial protection following WNV challenge. Better results were obtained for both vaccines after a third dose, i.e. alleviation of clinical signs, absence of fatalities and reduction of virus shedding and viraemia. Therefore the consequences of WNV infections in falcons can be clearly alleviated by vaccination, especially if the amended triple administration scheme is used, although side effects at the vaccination site must be accepted.

The use of semen evaluation and assisted reproduction in Spix's macaws in terms of species conservation

The Spixs macaw (Cyanopsitta spixii) is the rarest parrot on earth. The remaining captive population consists of 79 individuals. Captive propagation is ongoing to increase the number of individuals for future reintroduction back into the wild. Unfortunately, from 2004 to 2012, only 33 chicks hatched from 331 eggs. Semen evaluation and assisted reproduction might help to overcome this problem. Therefore, a recently developed electro-stimulated semen collection technique was used in Spixs macaws. Semen collection was successful in 39 of 78 attempts in 10 out of 17 males. Examination of the semen included evaluation of volume, color, consistency, contaminations and pH, as well as determination of motility, viability, morphology, concentration, and total count of spermatozoa. The median volume of semen samples was 5.6 µl. On average, 34.7 ± 21.9% (median 30%) of the sperm were motile and 23.1 ± 22.1% (median 16.5%) were progressively motile. In addition to spermatozoa, round cells were detected in the samples. Median sperm concentration was 15,500/µl (range 500-97,500/µl) and median viability was 50% (range 5-87%). Morphological examination revealed in 26.5% normal spermatozoa, high numbers of malformations of the head (50.2%) and tail region (20.5%), with 29% of all sperm showing multiple abnormalities. Artificial insemination was performed in three females; two eggs laid after artificial insemination had spermatozoa present on the perivitelline layer, suggesting the possible success of the insemination technique. Although no fertilization could be demonstrated, these preliminary results are promising, as they indicate that assisted reproduction might be a tool for species conservation in the Spixs macaw.

The role of 'atypical' Brucella in amphibians: Are we facing novel emerging pathogens?

To discuss together the novel cases of Brucella infections in frogs with the results of published reports to extend our current knowledge on ‘atypical’ brucellae isolated from amphibians and to discuss the challenges we face on this extraordinary emerging group of pathogens.

Vertebral Osteomyelitis and Septic Arthritis Associated With Staphylococcus hyicus in a Juvenile Peregrine Falcon (Falco peregrinus)

Abstract: A 6-week-old, parent-reared peregrine falcon (Falco peregrinus) was presented with spastic hypertonus of its hind limbs of unknown origin and duration. Radiologic examination revealed smooth periosteal reactions ventrally at thoracic vertebrae 5 to 7. Contrast-enhanced computed tomography identified the swelling as inflammation; antibiotic, antimycotic, anti-inflammatory, and analgesic treatments were initiated, and vitamins and minerals were supplemented. Because the birds condition did not improve after 10 days, it was euthanatized and submitted for postmortem examination. On histopathologic examination, chronic, active osteomyelitis was diagnosed in thoracic vertebrae 5 to 7, and chronic, active arthritis was present in both the right shoulder and left elbow joints. Staphylococcus hyicus was isolated from these 3 locations, as well as from lungs and liver, indicating a chronic septic staphylococcosis. Although infections with Staphylococcus species are occasional causes of vertebral osteomyelitis in juvenile poultry with active growth plates, it is only sporadically reported in raptors and companion birds. This case report is the first description of the clinical features and diagnostic and pathologic findings in a juvenile peregrine falcon with hematogenous osteomyelitis and arthritis associated with septicemia caused by S hyicus.

Single tracheal inoculation of Aspergillus fumigatus conidia induced aspergillosis in juvenile falcons (Falco spp.)

ABSTRACT Aspergillosis is a common and life-threatening respiratory disease in raptors with acute and chronic courses. Among raptors, gyrfalcons (Falco rusticolus) and their hybrids are often declared to be highly susceptible with juvenile individuals being the most susceptible. However, species- and age-specific experimental studies are lacking and minimal infective doses (IDs) for Aspergillus spp. conidia are unknown.Therefore, 8-week-old, healthy gyr-hybrid falcons (F. rusticolus X F. cherrug) (N = 18) were experimentally infected with Aspergillus fumigatus using a single intratracheal inoculation with varying dosages of conidia (102 to 107 conidia). Over 28 days, clinical signs were monitored as well as haematological and serological parameters. Following euthanasia, necropsy, histopathology, bacteriology, and mycology were performed. Re-isolated fungi were compared to the inoculum using microsatellite length polymorphisms. During the trial, clinical signs and dyspnoea correlated significantly with the ID. Necropsy revealed fungal lesions in the upper and lower airways of 10/18 inoculated falcons, but not in the control birds. In 9/18 inoculated falcons, fungal granulomas were confirmed in histopathology and A. fumigatus was re-isolated from these granulomas. Except one nasal isolate all re-isolated fungal strains were identical to the inoculum strain. Based on mycology and histopathology a minimal ID of 50% was calculated to be MID50% (±S.E.) = 104.52±0.67 for a single tracheal inoculation of A. fumigatus conidia. This study demonstrates for the first time that a single exposure is able to cause acute aspergillosis in juvenile falcons.