Mycoplasma hafezii sp. nov., isolated from the trachea of a peregrine falcon (Falco peregrinus).

Mycoplasma species are well known pathogens in avian medicine, especially in poultry. However, several Mycoplasma species have been regularly found in the respiratory tract of birds of prey which seem to be commensals in these bird species. In previous studies, an unknown Mycoplasma species which caused false positive results in a Mycoplasma meleagridis-specific PCR, was isolated from a tracheal swab of a clinically healthy, captive, adult peregrine falcon (Falco peregrinus). The isolate appeared in typical fried-egg-shaped colonies on SP4 agar plates and was dependent on sterol for growth. Acid was produced from glucose, but no arginine or urea was hydrolysed. The temperature range for growth was 28-44 °C, with an optimum at 37 °C. Strain M26T was serologically distinct from all species of the genus Mycoplasma with 16S rRNA gene sequence similarity ≥94 %. Biochemical, serological and molecular biological properties demonstrate that this organism represents a novel species of the genus Mycoplasma, for which the name Mycoplasma hafezii sp. nov. is proposed; the type strain is M26T (NCTC 13928, DSM 27652).

Lactococcus lactis, causative agent of an endocarditis valvularis and parietalis thromboticans in the allis shad, Alosa alosa (L.).

Since the 1940s, the anadromous allis shad, Alosa alosa (L.), has suffered population declines throughout its distribution range in Europe. In context of EU-LIFE projects for the reintroduction of the allis shad in the Rhine system, a comprehensive study was started in 2012 to investigate infectious diseases occurring in allis shad. In course of the study, 217 mature and young-of-the-year allis shad originating from the wild population from the Gironde-Garonne-Dordogne system (GGD-system) and the Rhine system as well as 38 allis shad from the breeding population were examined by use of bacteriological and histological methods. In 2012 and 2014, an endocarditis valvularis thromboticans caused by a coccoid bacterium was detected in 16% and 25% of mature allis shad originating from the GGD-system. Results of microbiologic examinations, including biochemical characteristics, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and 16S rRNA gene sequence analysis, revealed Lactococcus lactis as causative agent of this infection. This is the first report of an endocarditis valvularis and parietalis thromboticans caused by Lactococcus lactis in fish. Possible sources of infection as well as the impact for the reintroduction programme are discussed.

Investigation into diseases in free-ranging ring-necked pheasants (Phasianus colchicus) in northwestern Germany during population decline with special reference to infectious pathogens.

The population of ring-necked pheasants (Phasianus colchicus) is decreasing all over Germany since the years 2008/2009. Besides impacts of habitat changes caused by current rates of land conversion, climatic influences or predators, a contribution of infectious pathogens needs also to be considered. Infectious and non-infectious diseases in free-living populations of ring-necked pheasants have been scarcely investigated so far. In the present study, carcasses of 258 deceased free-ranging pheasants of different age groups, predominantly adult pheasants, collected over a period of 4 years in the states of Lower Saxony, North Rhine-Westphalia and Schleswig-Holstein, were examined pathomorphologically, parasitologically, virologically and bacteriologically, with a focus set on infectious pathogens. A periocular and perinasal dermatitis of unknown origin was present in 62.3% of the pheasants. Additional alterations included protozoal cysts in the skeletal musculature (19.0%), hepatitis (21.7%), enteritis (18.7%), gastritis (12.6%), and pneumonia (11.7%). In single cases, neoplasms (2.6%) and mycobacteriosis (1.7%) occurred. Further findings included identification of coronaviral DNA from trachea or caecal tonsils (16.8%), siadenoviral DNA (7.6%), avian metapneumoviral RNA (6.6%), and infectious bursal disease viral RNA (3.7%). Polymerase chain reaction (PCR) on herpesvirus, avian influenza virus (AIV), paramyxovirus type 1 (PMV-1), avian encephalomyelitis virus (AEV), and chlamydia were negative. Based on the present results, there is no indication of a specific pathogen as a sole cause for population decline in adult pheasants. However, an infectious disease can still not be completely excluded as it may only affect reproduction effectivity or a certain age group of pheasants (e.g., chicks) which were not presented in the study.

First report of a cystic malformation on the upper jaw of hatchery-reared allis shad Alosa alosa.

The anadromous allis shad Alosa alosa has suffered dramatic population declines throughout Europe and is currently considered as endangered throughout its entire distribution range. In order to reestablish allis shad in the River Rhine, which formerly housed one of the largest and most important populations, an EU-LIFE Project 'The re-introduction of allis shad in the Rhine system' was started in 2007. In course of the LIFE+ Projects, allis shad larvae bred from genitor fish of the Gironde-Garonne-Dordogne population in France were reared in a pilot ex situ stock plant pilot facility in Aßlar, Germany. At an age of 1-2 months, about 100% of these fish developed approximately 0.5- to 0.8-cm large, fluid-filled, transparent cysts in conjunction with the upper jaw. The performed microbiological, virological, parasitological and histological examinations did not detect any infectious agents. Possible causative agents are discussed with regard to environmental factors and the nutrition of larvae. In conclusion, the observed malformations are considered a sign for a severe health problem and therefore a risk for the successful breeding of allis shad in aquaculture.

Toltrazuril does not show an effect against pigeon protozoal encephalitis.

The protozoan parasite Sarcocystis calchasi causes a severe neurologic disease in domestic pigeons (Columba livia f. dom.) named pigeon protozoal encephalitis. Recently, the parasite has also been reported in psittacines causing a virtually identical disease with fatal outcome. So far, an etiological treatment of S. calchasi infections in pigeons or psittacines is unknown. The present study evaluates the effectiveness of the anticoccidian drug toltrazuril against S. calchasi and the influence of the timepoint of treatment. Therefore, nine domestic pigeons were inoculated with 400 S. calchasi sporocysts and treated with toltrazuril (25 mg/kg) in groups of three pigeons each at dpi 10/11 and dpi 40/41 and on two consecutive days at the onset of neurologic signs. After euthanasia at dpi 73, tissue samples including brain and skeletal muscles were examined by histology and S. calchasi-specific real-time PCR. All pigeons independent of the group developed neurologic signs from dpi 49 onwards. Histology identified sarcocysts in the skeletal muscles and a granulomatous encephalitis in the brains. The relative amount of S. calchasi DNA was on a comparable level in all pigeons. Consequently, toltrazuril was demonstrated to be not effective against S. calchasi with the applied treatment regime. Longer treatment periods or agents other the toltrazuril may be considered for further investigations. So far, preventive measures like roofing of aviaries for prevention of infection and regular disinfection remain the most important factor in the control of S. calchasi infections.

Comparison of diagnostic tools for the detection of aspergillosis in blood samples of experimentally infected falcons.

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 x 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 ß globulins, decreased percentages of prealbumin and albumin, and increased percentages of alpha 2 and ß globulins compared to inoculated falcons without granulomas. In conclusion, acute-phase proteins and the electrophoretic profile of birds challenged with A. fulmigatus show significant alterations, which in combination with other diagnostic procedures, assist in the early diagnosis of avian aspergillosis.

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 (10(7), 10(5) and 10(3)). 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 10(7) 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.

Unusual biphasic disease in domestic pigeons (Columba livia f. domestica) following experimental infection with Sarcocystis calchasi.

A novel Sarcocystis species has recently been reported in the domestic pigeon (Columba livia f. domestica) as intermediate host, causing severe central nervous signs similar to Paramyxovirus-1 or Salmonella Typhimurium var. cop. infection. Transmission of the parasite via the northern goshawk (Accipiter gentilis) as definitive host has been established. Experimental infection of domestic pigeons with sporocysts excreted by experimentally infected northern goshawks reproduced the natural infection in the pigeon, proving the causative role of the parasite in the disease. Here, we describe in greater detail the course of the fulminant biphasic disease depending on the infectious dose. Pigeons infected with 10(3) or 10(4) sporocysts showed clinical signs of polyuria and apathy around 10-11 days postinfection (dpi) and sudden neurological signs 51-57 dpi as a second phase of disease. Pigeons infected with higher doses died within 7-12 dpi, also showing polyuria and apathy but without nervous signs. At necropsy, livers and spleens had multifocal necroses and infestations with parasitic stages, namely, schizonts. Moreover, lesions and schizonts were also found in the lung, bone marrow, and next to blood vessels in the connective tissue of various organs. Pigeons infected with 102 sporocysts remained symptomless until 58-65 dpi, when sudden central nervous signs occurred. Major histopathologic findings of pigeons with neurological signs were encephalitis and myositis of virtually every skeletal muscle with high infestations of sarcocysts. Only mild myocarditis and very few cysts were found in the heart muscles. Importantly, a sentinel pigeon developed identical lesions when compared to those of low-dose infected pigeons, suggesting a risk of mechanical transmission of sporocysts from freshly infected to uninfected pigeons in a flock. By contrast, chickens failed to develop any clinical signs or pathologic lesions in the same experiment. The findings further characterize the new highly pathogenic disease in domestic pigeons, which clinically mimics paramyxovirosis and salmonellosis in both phases of the disease and exclude chickens as further intermediate host species.

[Recommendations for the veterinary care and assessment of bird of prey collections]. / Empfehlungen für die tierärztliche Bestandsbetreuung und die Beurteilung von Greifvogelhaltungen.

Legislation from a new regulation of the Federal Nature Conservation Act that became effective on March 1, 2010 requires a written program for veterinary prophylaxis, treatment and nutrition for zoo and animal collections. As a result of this act, veterinary care is now obligatory for all captive birds of prey kept within either private or commercial collections, independent on the number of birds involved. The legal requirements of the Act will shortly be introduced and recommendations for the veterinary care of bird of prey collections are provided. Firstly, risk assessment of different husbandry systems (falconry birds, show birds, breeding stock, rehabilitation) is performed and veterinary care programs are provided based upon these assessments. Additionally, instructions for anamnestic ascertainments, work flow, feeding, quarantine, cleaning and disinfection procedures as well as prophylactic measures such as vaccination are provided. As husbandry, in particular the size and equipment of cages is important for the health and welfare of the birds, species specific housing, care and protection measures are also discussed. Veterinarians will be able to refer to these guidelines and can use them in the future for collection management.

Central nervous signs, blindness and cerebral vermicosis in free-ranging peregrine falcons (Falco peregrinus) associated with aberrant larval migrations.

Four free-ranging peregrine falcons (Falco peregrinus) were submitted with a history of unilateral or bilateral blindness and central nervous signs to a veterinary clinic in Germany. There were no indications of trauma or ocular disease. Likewise, other differential diagnoses for CNS signs were ruled out within the diagnostic process. The clinical diagnostic panel in live falcons included general examination, radiography, endoscopy, hematology, ophthalmoscopy and parasitological examination of the feces, blood gas analysis and blood chemistry as well as computed tomography, and magnetic resonance imaging (MRI). A complete pathological and histopathological examination was performed post-mortem. The only common finding in all birds was an infection with the nematode parasite Serratospiculum tendo. The parasite was confirmed morphologically and via PCR. In two falcons intracerebral vermicoses was suspected in MRI and confirmed in subsequent histopathology, but molecular biological identification of the parasite species failed from brain tissue. Until today, S. tendo had been reported to affect the respiratory system, the liver and different parts of the gastrointestinal tract and to cause cachexia, inappetence, regurgitation, dyspnea and general signs of illness such as lethargy, poor plumage, and reduced reproduction. Our findings indicate that aberrant migration should be considered as cause for CNS signs in falcons. As S. tendo might be a possible cause for this, CNS signs might be included in the list of clinical signs of serratospiculiasis in falcons.

Semen analysis and successful artificial insemination in the St. Vincent amazon (Amazona guildingii).

The St. Vincent amazon (Amazona guildingii) is an endemic parrot on the Carribean island St Vincent. Due to poaching, trade, natural events such as hurricanes and habitat loss the species declined severely throughout the 20th century to a total number of 487 adult individuals and is currently regarded as vulnerable by IUCN. Captive breeding is attempted in terms of species conservation, but reproduction rates have been low due to reproductive problems such as mate aggression, asynchronous reproductive activity and infertile eggs. The aims of the present study were; firstly, to evaluate whether semen analysis might help to assess the fertility of males and to detect potential reasons for infertile eggs; secondly, to increase the number of offspring using artificial insemination, and as a future effect, to increase the presence of genetically valuable males in the ex-situ breeding population. For semen collection electric stimulation was used in 15 mature and healthy St. Vincent amazons with a success rate of 89% (202/227 attempts) in 14/15 males. Quality assessment of the semen included the evaluation of volume, pH, color, consistency and contaminations of the ejaculate, as well as estimation of motility, viability, morphology, concentration and total count of spermatozoa. Semen pH ranged from 6.7 to 7.5. Median sperm motility was 50% and median progressively forward motility 40%. Mean sperm concentration (x¯ ± SD) was 21,313.5 ± 22,408.8 spermatozoa/µl and mean sperm viability 66 ± 16%. Semen samples contained on average 20.5% morphologically normal spermatozoa and sperm malformations were detected mainly in the head (x¯ = 47.6%) and the tail regions (x¯ = 27.7%). Interestingly round bodies were detected in the ejaculates with a mean ratio of 0.6 round bodies per sperm. Semen analysis proved to be very useful to identify males with poor semen quality. Artificial insemination was performed 46 times in 9 females with either individual or pooled semen samples and 13 eggs from 7 females were laid afterwards. In 3 eggs, embryonic development was detected and 1 chick hatched successfully. Paternity testing confirmed the fatherhood of a one-winged semen donor male, a bird which was not able to copulate naturally. The results are very promising and underline that assisted reproduction techniques are a suitable tool for species conservation in captive breeding programs for psittacines.

Health status of free-ranging ring-necked pheasant chicks (Phasianus colchicus) in North-Western Germany.

Being a typical ground-breeding bird of the agricultural landscape in Germany, the pheasant has experienced a strong and persistent population decline with a hitherto unexplained cause. Contributing factors to the ongoing negative trend, such as the effects of pesticides, diseases, predation, increase in traffic and reduced fallow periods, are currently being controversially discussed. In the present study, 62 free-ranging pheasant chicks were caught within a two-year period in three federal states of Germany; Lower Saxony, North Rhine-Westphalia and Schleswig-Holstein. The pheasant chicks were divided into three age groups to detect differences in their development and physical constitution. In addition, pathomorphological, parasitological, virological, bacteriological and toxicological investigations were performed. The younger chicks were emaciated, while the older chicks were of moderate to good nutritional status. However, the latter age group was limited to a maximum of three chicks per hen, while the youngest age class comprised up to ten chicks. The majority of chicks suffered from dermatitis of the periocular and caudal region of the head (57-94%) of unknown origin. In addition, intestinal enteritis (100%), pneumonia (26%), hepatitis (24%), perineuritis (6%), tracheitis (24%), muscle degeneration (1%) and myositis (1%) were found. In 78% of the cases, various Mycoplasma spp. were isolated. Mycoplasma gallisepticum (MG) was not detected using an MG-specific PCR. Parasitic infections included Philopteridae (55%), Coccidia (48%), Heterakis/Ascaridia spp. (8%) and Syngamus trachea (13%). A total of 8% of the chicks were Avian metapneumovirus (AMPV) positive using RT-PCR, 16% positive for infectious bronchitis virus (IBV) using RT-PCR, and 2% positive for haemorrhagic enteritis virus (HEV) using PCR. All samples tested for avian encephalomyelitis virus (AEV), infectious bursal disease virus (IBDV) or infectious laryngotracheitis virus (ILTV) were negative. The pool samples of the ten chicks were negative for all acid, alkaline-free and derivative substances, while two out of three samples tested were positive for the herbicide glyphosate. Pheasant chick deaths may often have been triggered by poor nutritional status, probably in association with inflammatory changes in various tissues and organs as well as bacterial and parasitic pathogens. Theses impacts may have played a major role in the decline in pheasant populations.

A novel Sarcocystis-associated encephalitis and myositis in racing pigeons.

Sarcosporidian cysts in the skeletal muscle of domestic pigeons (Columba livia f. domestica) have previously been attributed to infection with Sarcocystis falcatula, which is shed in the faeces of the opossum (Didelphis virginiana). Here, we describe fatal spontaneous encephalitis and myositis associated with Sarcocystis infections in three flocks of racing pigeons with 47 of 244 animals affected. The clinical course was characterized by depression, mild diarrhoea, torticollis, opisthotonus, paralysis and trembling. Histopathological examination of 13 pigeons revealed generalized severe granulomatous and necrotizing meningoencephalitis and myositis with sarcosporidian cysts. Light and transmission electron microscopy identified cysts in heart and skeletal muscle of 1 to 2 mm in length and 20 to 50 microm in width. These were subdivided into small chambers by fine septae and filled with lancet-shaped cystozoites (7.5 x 1.5 microm) and dividing metrocytes, which is characteristic for Sarcocystis. The cysts had smooth walls and were devoid of protrusions typical of S. falcatula. Polymerase chain reaction amplification and sequencing of the internal transcribed spacer region (ITS-1) and the complete 28S rRNA identified a novel Sarcocystis species with only 51% ITS-1 nucleotide sequence similarity with S. falcatula. A phylogenetic comparison of the 28S rRNA revealed close sequence homologies with Frenkelia microti, Frenkelia glareoli and Sarcocystis neurona. The clinical, histopathological, electron microscopic and genetic data are unlike any previously described protozoan infections in pigeons, suggesting a novel, severe disease due to an as yet undescribed Sarcocystis species.

The role of Virus "X" (Tortoise Picornavirus) in kidney disease and shell weakness syndrome in European tortoise species determined by experimental infection.

Tortoise Picornavirus (ToPV) commonly known as Virus "X" was recently discovered in juvenile European tortoises suffering from soft carapace and plastron as well as kidney disease. Therefore, this virus was a potential candidate to be a causative agent for these disease patterns. Spur thighed tortoises (Testudo graeca) seemed to be more susceptible to establish clinical symptoms than other European species like T. hermanni. Thus this trial investigated the role of ToPV in the described syndrome. Two groups of juvenile European tortoises (T. graeca and T.hermanni) each of 10 animals, were cloacally, oronasally and intracoelomically inoculated with an infectious dose (~ 2000 TICD) of a ToPV strain isolated from a diseased T. graeca. A control group of two animals of each species received non-infected cell culture supernatant. The tortoises were examined daily and pharyngeal and cloacal swabs for detection of ToPV-RNA by RT-PCR were taken from each animal every six days for a period of 6 months. At the end of the study the remaining animals were euthanised and dissected. Bacteriological and parasitological tests were performed and organ samples of all tortoises were investigated by RT-PCR for the presence of ToPV and histopathology. Animals that were euthanised at the end of the experiment, were examined for presence of specific anti-ToPV antibodies. Several animals in both inoculated groups showed retarded growth and a light shell weakness, in comparison to the control animals. Three animals were euthanised during the trial, showing reduced weight gain, retarded growth, severe shell weakness and apathy, in parallel to clinical observations in naturally infected animals. In all inoculated animals of both species an intermittent virus shedding, starting from 18 days post inoculation (d.p.i.), till 164 d.p.i. was detected, while the control animals remained negative. The virus was successfully reisolated in terrapene heart cell culture in 16 of 20 inoculated animals of both species. Histopathology of most inoculated animals revealed a lack of bone remodeling and vacuolisation in kidney tubuli which supports the described pathogenesis of nephropathy and osteodystrophy. Anti- ToPV antibody titres ranged from 1:2 to >1:256 in 13 of 20 animals, whereas all control animals were seronegative. The study proofed the Henle Koch`s postulates of ToPV as causative agent for shell dystrophy and kidney disease in both testudo species. The proposed species specific sensitivity towards clinical disease was not observed.

Semen collection, semen analysis and artificial insemination in Columbian sharp-tailed grouse (Tympanuchus phasianellus columbianus) as part of a species conservation project.

Columbian sharp-tailed grouse (Tympanuchus phasianellus columbianus; hereafter CSTG) have experienced substantial decreases in population numbers and geographic range during the early 20th century, primarily due to habitat loss. The conservation aim of this project was to re-establish a self-sustaining population of CSTG within an unoccupied portion of their historic range in northeastern Nevada via reintroduction from source populations in Idaho, USA. Female nest initiation rates post-translocation due to low fertilization rates are believed to be one limiting factor in the establishment of some translocated CSTG populations. However, studies on semen collection and artificial insemination in this species are absent. Assisted reproduction was evaluated as an additional tool in this species conservation project in order to gain knowledge on the reproductive status of yearling and adult male CSTG, establish orientation values for semen parameters and evaluate artificial insemination procedures on female CSTG. In two consecutive breeding seasons, semen collection was attempted 51 times in 47 males using the established massage method, and a novel electro-stimulation technique. Semen collection was successful in all attempts, even in yearling grouse, which represents a novel confirmation that yearling male CSTG can produce live spermatozoa in their first breeding season. Volume, color, consistency, contamination, pH of semen, and the motility, concentration, viability and morphology of spermatozoa were analyzed. Extracted semen volume ranged between 6 and 74 µl and the mean pH was 6.9 ±â€¯0.5 (x¯ ± SD). Morphology analysis revealed that on average, 42.8% of sperm was morphologically normal, but 34.8% had malformed tails. Additionally, artificial insemination was practiced in 17 females (sham-insemination group; insemination lacking spermatozoa) and performed in 17 females. Intravenous catheters G20 1.0 mm × 32 mm enabled safe intravaginal insemination under visual control. Significant (p ≤ 0.05) differences in semen parameters between adult and yearling birds were detected. It is well established that adult males receive a majority of copulations during lekking, but our novel findings also indicate that they produce significantly more spermatozoa which is of higher quality than yearling males. This finding offers insights into male reproductive biology in a lekking grouse species.

Occurrence of mycoplasmas in semen samples of birds of prey.

Mycoplasmas are well-known pathogens in a variety of animals. In poultry it is known that some species can be transmitted by semen and infect the uterus of females. As the prevalence of mycoplasmas in birds of prey is very high and artificial insemination is a commonly used technique for reproduction, the possibility of transmission Mycoplasma spp. by contaminated semen in birds of prey was investigated. Isolation of mycoplasmas was possible in five out of 32 (15.6%) semen samples of different bird of prey species. Two additional semen samples were positive for mycoplasma DNA using a Mycoplasma-genus-specific polymerase chain reaction. The isolation of mycoplasmas from a testicular sample indicates the testis as the possible source of contamination. Sequencing of large parts (>90%) of the 16S rRNA gene of the isolated mycoplasmas suggests that all isolates belong to the same species. Alignment of the sequenced products with the 16S rRNA gene of Mycoplasma species in GenBank demonstrated a similarity of 97% to Mycoplasma verecundum, but serological testing by immunobinding assay failed to identify it as such. It is recommended that the semen of donor birds of prey is examined for mycoplasmas before its use in artificial insemination.

Use of polymerase chain reactions to detect Mycoplasma gallisepticum, Mycoplasma imitans, Mycoplasma iowae, Mycoplasma meleagridis and Mycoplasma synoviae in birds of prey.

Certain Mycoplasma spp. are pathogens of poultry, but little is known of the role of mycoplasmas in disease of birds of prey. Species-specific polymerase chain reactions (PCRs) for the detection of the poultry pathogens Mycoplasma gallisepticum, Mycoplasma imitans, Mycoplasma iowae, Mycoplasma meleagridis and Mycoplasma synoviae were therefore evaluated for use in birds of prey. The specificities of the PCR methods were established using avian and other mycoplasmas and also selected walled bacteria. The sensitivities of the different PCR assays varied between 100 fg and 10 pg DNA. Fifty-three tracheal swabs from healthy captive and free-ranging birds of prey were then investigated using these PCRs, and in no case was an amplicon obtained for M. gallisepticum/M. imitans, M. iowae or M. synoviae. Species-specific primers for M. meleagridis amplified a product from eight birds of prey but restriction enzyme analysis as well as sequencing of PCR products demonstrated these results to be false positives. Alignment studies of the sequenced products with the 16S rRNA gene sequence of various Mycoplasma species in GenBank demonstrated an identity of 91% to M. meleagridis but of 98% to Mycoplasma buteonis or Mycoplasma gallopavonis. Isolation and attempted identification of these mycoplasmas suggested it may be a previously unrecognized species.

The role of mycoplasmas in a conservation project of the lesser kestrel (Falco naumanni).

The lesser kestrel (Falco naumanni) is one of the most endangered bird species in Europe, and a captive breeding and reintroduction project was established. A breeding project is vulnerable to pathogens, e.g., mycoplasmas, reducing the reproductive success and carrying the risk to release pathogens with the birds to the wild. Therefore, 18 infertile eggs and 43 dead in shell embryos of the breeding project, as well as 27 nestlings and 34 adult birds of the captive and three different free-ranging populations were investigated for the occurrence of mycoplasmas by culture and a Mycoplasma genus-specific polymerase chain reaction. All eggs, embryos, and hand-reared nestlings from the captive group were negative. In contrast, all parent-reared nestlings and 88% of the adults were positive. Mycoplasma falconis and unidentifiable mycoplasmas were detected in all groups. Mycoplasma buteonis was found in the captive and only in two of the three free-ranging populations. Sequencing the 16S rRNA gene of six randomly selected unidentified isolates showed that five isolates were similar and most likely had been found previously in a falcon from Germany. The remaining isolate demonstrated a very high homology to unidentified Mycoplasma isolates obtained previously from semen samples of raptors. The results suggest that these isolates might represent two new species. Mycoplasmas seem not to play a major role as pathogens in the breeding project, and there is no evidence that releasing birds poses a risk to the free-ranging population with regard to mycoplasmas. The study seems to be the first to describe the occurrence and role of mycoplasmas in the lesser kestrel.

Occurrence of mycoplasmas in free-ranging birds of prey in Germany.

Mycoplasmas are well-known avian pathogens of poultry and some passerines. Although reported in birds of prey, their role as pathogens is still unclear. Healthy, free-ranging raptor nestlings sampled during a routine ringing (banding) program, and birds of prey from rehabilitation centers, tested positive for Mycoplasma spp. by culture and a genus-specific polymerase chain reaction (PCR). Given the lack of clinical signs and disease, we suggest that mycoplasmas in raptors may be commensal rather than pathogenic. Using immunobinding assay and species-specific PCR tests, Mycoplasma buteonis, M. falconis, and M. gypis were identified; M. falconis was only detected in falcons. Additionally, some isolates could not be identified. This is the first report of Mycoplasma spp. isolations from Western Marsh Harriers (Circus aeroginosus), a Eurasian Hobby (Falco subbuteo), and a Barn Owl (Tyto alba).

Clinical signs and diagnosis of thiamine deficiency in juvenile goshawks (Accipiter gentilis).

Between 2002 and 2006, a large number of juvenile northern goshawks (Accipiter gentilis) with central nervous signs were examined. They were aged between 45 and 55 days and had been fed on frozen and thawed day-old chicks. High-performance liquid chromatography showed that the birds had whole blood thiamine levels between 2.2 and 6.0 microg/l; the concentrations of other blood constituents were within their reference ranges. Treatment with thiamine hydrochloride rapidly resolved the clinical signs. Measurements of the concentration of thiamine in 22 free-ranging and captive goshawks showed that they ranged from 45.1 to 200 microg/l.