First results from the releases of Cinereous Vultures (Aegypiusmonachus) aiming at re-introducing the species in Bulgaria - the start of the establishment phase 2018-2022.

The current work presents the preliminary results of the Cinereous Vulture (Aegypiusmonachus) releases in the Balkan Mountains in 2018-2022, aiming at the species re-introduction in Bulgaria, where it was listed as locally extinct since 1985. The first imports and releases of Cinereous Vultures in Bulgaria started in 2018. Until mid-2022, 72 individuals were released in the Eastern Balkan Mountains (Kotlenska Planina SPA and Sinite Kamani Nature Park) and Vrachanski Balkan Nature Park. Of them, 63 immatures imported from Spain were released from aviaries and nine juveniles captive-bred in European zoos were released by hacking (fledging from an artificial nest). We compared the success in survival and establishment between the different release sites and methods used to adjust the ongoing technics and tactics and to support knowledge improvement for future similar projects. From the nine Cinereous Vultures released by hacking, the results were as follows: 1.00 fledging success, but only 0.22 survival in the first six months - combined period of acclimation, first migration and the first winter. All survivors from that period reached maturity into the wild, but all emigrated from the release site and settled elsewhere. Of the 63 individuals released by aviaries, 32 individuals were released in the Eastern Balkan Mountains (18 individuals are still alive - 0.56 survival; 14 individuals settled in the area, which accounts for 0.44 of all released birds and 0.78 of the survivors). Thirty-one individuals were released in Vrachanski Balkan Nature Park (23 individuals are still alive - 0.74 survival; 22 individuals settled in the area - 0.71 of all released birds and 0.96 of the survivors). Based only on aviary method comparison, the settling of the individuals in the release area was alike in the two sites. However, the Vrachanski Balkan Nature Park performed better in survival - both in acclimation and establishment periods. While comparing the release methods - hacking and release from the aviary - the following results were observed: the survival rate during acclimation was 0.86. Due to more considerable losses during the first migration and dispersal in the individuals released by hacking, the survival rate of 0.22 was significantly lower compared to 0.73 for the birds released from the aviary. Additionally, in both methods, a similar pattern in the first winter and spring migration dispersal was observed. Although the survival was equal in the released-by-hacking or aviary birds after the first year onwards, it is essential to note that the emigration of the hacked birds from the release site was 1.00. In comparison, the birds released from aviaries largely remained and settled in the release area (> 0.77 of the survivors). The cost of release and related acclimation, settling, dispersal and the first winter was the greatest: 0.12-0.17 per period, or cumulatively, it was about 0.27. Survival increased and stabilised to > 0.90 after the first year in the wild and reached nearly 1.00 after two years in the wild onwards. Two distinct nuclei of the Cinereous Vulture were established along the Balkan Mountains - the Eastern Balkan Mountains with 18-23 individuals and four formed pairs using a territory of about 642.74 km2 - 95% home range and 85.72 km2 - 50% core area with center being the town of Kotel; and Vrachanski Balkan Nature Park with present 23-29 individuals, of which 2-3 pairs formed so far, using a territory of about 1,143.66 km2 - 95% home range and 22.89 km2 - 50% core area with center being the village of Zgorigrad. The species readily accepted breeding in artificial nest platforms built by professional arborists on different tree species - oak, beech, sycamore and pine. The only naturally built nests were on the ground (n = 2) (unsuccessful) and in Scots Pine (n = 1) (successful). In 2021 and 2022, in each of the two sites, the first successful reproductions were recorded, which marked the return of the Cinereous Vulture as breeding species - 28 years after the last occasional record of a single breeding pair in the country and 36 years after it was officially listed as locally extinct in Bulgaria.

Results of the re-introduction of the Griffon Vulture (Gypsfulvus) in Vrachanski Balkan Nature Park, Bulgaria - completion of the establishing phase 2010-2020.

The current study analyses and presents the results of the ten-year establishment phase of the Griffon Vulture (Gypsfulvus) local re-introduction in Vrachanski Balkan Nature Park, north-western Bulgaria. Between 2010 and 2020, 61 rehabilitated and captive-bred Griffon Vultures from Spain, France and several European zoos were released from an acclimatisation aviary. The first successful breeding in the wild was reported in 2015. Thus, the species has been restored as a nesting species in the area after more than 60 years of absence. In 2020, the local population accounted for some 55-70 individuals, consisting of about 20-23 breeding pairs in three-five separate colonies and two frequently-used roosting sites. Forty-two chicks fledged from 2010 to 2020, at an average breeding success of 0.46 chicks/territorial pair and productivity of 0.62 fledglings/breeding pair. The mortality rate is calculated at 0.34; an additional 0.07 of the released individuals have never been seen or found. The local nucleus of the Griffon Vulture now covers a territory of 1,478.58 km², calculated as a 95% home range, while the 50% core area is 9.07 ± 5.73 km2 (range 2.12-22.89 km2). With these results, we consider the establishment phase of the re-introduction of the species in Vrachanski Balkan Nature Park as completed.

New insight into spatial ecology of Griffon Vulture (Gypsfulvus) on the Balkans provides opportunity for focusing conservation actions for a threatened social scavenger.

The knowledge in the behaviour and movement of endangered species is of key importance for the precise targeting and assessing the efficiency of nature conservation actions, especially considering vultures, which explore vast areas to locate ephemeral and unpredictable food resources. Therefore, a total of 51 Griffon Vultures (Gypsfulvus) from both the re-introduced population and the autochthonous Balkan Peninsula (Balkans) colonies have been tagged with GPS/GSM transmitters in recent years, in order to study their seasonal and spatial distribution. The current study presents the analysis of the high-resolution GPS location data, acquired between January 2016 and March 2021. A total of 1,138,383 locations (an average number of 23,716 ± 18,886 positions per bird, ranged between 2,515 and 76,431 of total fixes per bird; n=48) were used to estimate the home range size and identify the traditional foraging areas and roosting sites of the birds during the wintering, migration/roaming and summering periods. Our results reveal that Griffon Vultures movement activity and home range size varied considerably throughout the annual cycle, especially between their wintering and summering grounds, while exhibiting significant overlapping amongst the tracked individuals. Specifically, immature Griffon Vultures travel long distances across all Balkan Peninsula countries, but always gather with conspecifics, showing strong fidelity to active breeding/roosting sites. The total home range 95% area of the Griffon Vulture population on the Balkans was estimated at 39,986.4 km² and the 50% core area at 1,545.42 km² (n = 48). All tracked birds were found to either visit or frequently use (> 95% of the time) the same seven vulture key zones on the Balkan Peninsula - one in Serbia, one shared between North Macedonia and Bulgaria, one shared between Bulgaria and Greece, two entirely lying in Bulgaria, one in western Greece and one shared between Kvarner Archipelago islands in Croatia and the Julian Alps - Italy, Austria and Slovenia. Several smaller sub-zones were also defined within these general ones. The seven key zones form a coherent network and are used as stepping stones for Griffon Vultures during their migration movements and roaming, but also wintering and summering. The observed concentration tendency of Griffon Vultures on the Balkans and the predictability of their temporal and spatial presence should be used to precisely target, address and substantially increase the efficiency of the conservation measures in this marginal and, thus, still vulnerable meta-population.

Variants in PRKAR1B cause a neurodevelopmental disorder with autism spectrum disorder, apraxia, and insensitivity to pain.

PURPOSE: We characterize the clinical and molecular phenotypes of six unrelated individuals with intellectual disability and autism spectrum disorder who carry heterozygous missense variants of the PRKAR1B gene, which encodes the R1ß subunit of the cyclic AMP-dependent protein kinase A (PKA). METHODS: Variants of PRKAR1B were identified by single- or trio-exome analysis. We contacted the families and physicians of the six individuals to collect phenotypic information, performed in vitro analyses of the identified PRKAR1B-variants, and investigated PRKAR1B expression during embryonic development. RESULTS: Recent studies of large patient cohorts with neurodevelopmental disorders found significant enrichment of de novo missense variants in PRKAR1B. In our cohort, de novo origin of the PRKAR1B variants could be confirmed in five of six individuals, and four carried the same heterozygous de novo variant c.1003C>T (p.Arg335Trp; NM_001164760). Global developmental delay, autism spectrum disorder, and apraxia/dyspraxia have been reported in all six, and reduced pain sensitivity was found in three individuals carrying the c.1003C>T variant. PRKAR1B expression in the brain was demonstrated during human embryonal development. Additionally, in vitro analyses revealed altered basal PKA activity in cells transfected with variant-harboring PRKAR1B expression constructs. CONCLUSION: Our study provides strong evidence for a PRKAR1B-related neurodevelopmental disorder.

In vitro and ex vivo studies on diltiazem hydrochloride-loaded microsponges in rectal gels for chronic anal fissures treatment.

Diltiazem hydrochloride, topically applied at 2% concentration, is considered effective for the treatment of chronic anal fissures, although it involves several side effects among which anal pruritus and postural hypotension. To test the hypothesis that a sustained delivery system of diltiazem hydrochloride may be helpful for the treatment of chronic anal fissures, in the present study we evaluated the potential of gels containing diltiazem hydrochloride entrapped in microsponges. Such microsponges were based on Eudragit RS 100 and the effect of some formulation variables was assessed by a 23 full factorial screening design. An optimized formulation of diltiazem hydrochloride microsponges was dispersed in Methylcellulose 2% or Poloxamer 407 20% and the resulting gels (micro-l-diltiazem hydrochloride 2%) were subjected to in vitro drug release, ex vivo permeability and drug deposition after application on porcine rectal mucosa. The results showed a prolonged release up to 24 h from micro-l-diltiazem hydrochloride at 2% in the gels. The permeation tests revealed up to 18% higher drug retention on the mucosal tissue after 24 h by the micro-l-diltiazem hydrochloride 2% gels compared to conventional diltiazem hydrochloride gels at 2%. These results suggest that diltiazem hydrochloride-loaded microsponges dispersed in rectal gels may be useful to overcome some limitations of conventional local chronic anal fissure therapy.

Blood parasite infections in a wild population of ravens (Corvus corax) in Bulgaria.

BACKGROUND: Blood parasites have been studied intensely in many families of avian hosts, but corvids, a particularly cosmopolitan family, remain underexplored. Haemosporidian parasites of the common raven (Corvus corax) have not been studied, although it is the largest, most adaptable, and widespread corvid. Genetic sequence data from parasites of ravens can enhance the understanding of speciation patterns and specificity of haemosporidian parasites in corvids, and shed light how these hosts cope with parasite pressure. METHODS: A baited cage trap was used to catch 86 ravens and a nested PCR protocol was used to amplify a 479 bp fragment of the haemosporidian cytochrome b gene from the samples. The obtained sequences were compared with the MalAvi database of all published haemosporidian lineages and a phylogenetic tree including all detected raven parasites was constructed. An examination of blood smears was performed for assessment of infection intensity. RESULTS: Twenty blood parasite lineages were recovered from ravens caught in a wild population in Bulgaria. The prevalence of generalist Plasmodium lineages was 49%, and the prevalence of Leucocytozoon lineages was 31%. Out of 13 detected Leucocytozoon lineages six were known from different corvids, while seven others seem to be specific to ravens. A phylogenetic reconstruction suggests that Leucocytozoon lineages of ravens and other corvids are not monophyletic, with some groups appearing closely related to parasites of other host families. CONCLUSIONS: Several different, morphologically cryptic groups of Leucocytozoon parasites appear to infect corvids. Ravens harbour both generalist corvid Leucocytozoon as well as apparently species-specific lineages. The extraordinary breeding ecology and scavenging lifestyle possibly allow ravens to evade vectors and have relatively low blood parasite prevalence compared to other corvids.