Juan Manuel Blanco

Species Variation in Osmotic, Cryoprotectant, and Cooling Rate Tolerance in Poultry, Eagle, and Peregrine Falcon Spermatozoa

Abstract Potential factors influencing spermatozoa survival to cryopreservation and thawing were analyzed across a range of the following avian species: domestic chicken (Gallus domesticus), domestic turkey (Meleagris gallopavo), golden eagle (Aquila chrysaetos), Bonellis eagle (Hieraaetus fasciatus), imperial eagle (Aquila adalberti), and peregrine falcon (Falco peregrinus). Studies focused on spermatozoa tolerance to the following: 1) osmotic stress, 2) different extracellular concentrations of the cryoprotectant dimethylacetamide (DMA), 3) equilibration times of 1 versus 4 h, 4) equilibration temperature of 4 versus 21°C, and 5) rapid versus slow cooling before cryopreservation and standard thawing. Sperm viability was assessed with the live/dead stain (SYBR-14/propidium iodine). Sperm viability at osmolalities ≥800 mOsm was higher (P < 0.05) in raptor than poultry semen. Return to isotonicity after exposure to hypertonicity (3000 mOsm) decreased (P < 0.05) number of viable spermatozoa in chicken, turkey, and golden and Bonellis eagle spermatozoa but not in imperial eagle or peregrine falcon spermatozoa. Differences were found in spermatozoa resistance to hypotonic conditions, with eagle species demonstrating the most tolerance. Semen, equilibrated for 1 h (4°C) in diluent containing DMA (≥2.06 M), experienced decreased (P < 0.05) spermatozoa survival in all species, except the golden eagle and peregrine falcon. Number of surviving spermatozoa diminished progressively with increasing DMA concentrations in all species. Increased equilibration temperature (from 4 to 21°C) markedly reduced (P < 0.05) spermatozoa survival in all species except the Bonellis eagle and turkey. Rapid cooling was detrimental (P < 0.05) to spermatozoa from all species except the imperial eagle and the chicken. These results demonstrate that avian spermatozoa differ remarkably in response to osmotic changes, DMA concentrations, equilibration time, temperature, and survival after fast or slow freezing. These differences emphasize the need for species-specific studies in the development and enhancement of assisted breeding for poultry and endangered species.

Pathogenicity of two recent Western Mediterranean West Nile virus isolates in a wild bird species indigenous to Southern Europe: the red-legged partridge

West Nile virus (WNV) is an emerging zoonotic pathogen whose geographic spread and incidence in humans, horses and birds has increased significantly in recent years. WNV has long been considered a mild pathogen causing self-limiting outbreaks. This notion has changed as WNV is causing large epidemics with a high impact on human and animal health. This has been particularly noteworthy since its introduction into North America in 1999. There, native bird species have been shown to be highly susceptible to WNV infection and disease with high mortalities. For this reason, the effect of WNV infection in North American bird species has been thoroughly studied by means of experimental inoculations in controlled trials. To a lesser extent, European wild birds have been shown to be affected clinically by WNV infection. Yet experimental studies on European wild bird species are lacking. The red-legged partridge (Alectoris rufa) is a gallinaceous bird indigenous to the Iberian Peninsula, widely distributed in South Western Europe. It plays a key role in the Mediterranean ecosystem and constitutes an economically important game species. As such it is raised intensively in outdoor facilities. In this work, red-legged partridges were experimentally infected with two recent WNV isolates from the Western Mediterranean area: Morocco/2003 and Spain/2007. All inoculated birds became viremic and showed clinical disease, with mortality rates of 70% and 30%, respectively. These results show that Western Mediterranean WNV variants can be pathogenic for some European bird species, such as the red-legged partridge.

Worldwide Phylogenetic Relationship of Avian Poxviruses

ABSTRACT Poxvirus infections have been found in 230 species of wild and domestic birds worldwide in both terrestrial and marine environments. This ubiquity raises the question of how infection has been transmitted and globally dispersed. We present a comprehensive global phylogeny of 111 novel poxvirus isolates in addition to all available sequences from GenBank. Phylogenetic analysis of the Avipoxvirus genus has traditionally relied on one gene region (4b core protein). In this study we expanded the analyses to include a second locus (DNA polymerase gene), allowing for a more robust phylogenetic framework, finer genetic resolution within specific groups, and the detection of potential recombination. Our phylogenetic results reveal several major features of avipoxvirus evolution and ecology and propose an updated avipoxvirus taxonomy, including three novel subclades. The characterization of poxviruses from 57 species of birds in this study extends the current knowledge of their host range and provides the first evidence of the phylogenetic effect of genetic recombination of avipoxviruses. The repeated occurrence of avian family or order-specific grouping within certain clades (e.g., starling poxvirus, falcon poxvirus, raptor poxvirus, etc.) indicates a marked role of host adaptation, while the sharing of poxvirus species within prey-predator systems emphasizes the capacity for cross-species infection and limited host adaptation. Our study provides a broad and comprehensive phylogenetic analysis of the Avipoxvirus genus, an ecologically and environmentally important viral group, to formulate a genome sequencing strategy that will clarify avipoxvirus taxonomy.

Comparative cryopreservation of avian spermatozoa: Benefits of non-permeating osmoprotectants and ATP on turkey and crane sperm cryosurvival

A comparative approach was used to evaluate the cryosurvival of turkey and crane sperm frozen in a dimethylacetamide (DMA) cryodiluent supplemented with osmoprotectants and ATP. A range (6-26%) of DMA concentrations was used alone or in combination with ATP (30, 60 or 118mM) or one of the following osmoprotectants: (1) sucrose (turkey, 8.0%; crane, 5.0%); (2) 5.0% sucrose and 5.0% trehalose; or (3) betaine hydrochloride (0.1, 0.2 or 0.4mM). The viability of thawed sperm was assessed using the nigrosin-eosin stain and sperm motility was determined using the hanging-drop technique. For semen frozen only with DMA, post-thaw sperm motility was greatest (P<0.05) for the 6.0%, 10.0% and 18% concentrations, regardless of species. Turkey sperm frozen with the sucrose/trehalose combination had greater (P<0.05) post-thaw motility for all DMA treatments compared to DMA alone. The lowest concentration of the osmoprotectant betaine hydrochloride substantially improved turkey sperm viability post-thaw in all treatments compared to DMA alone (P<0.05). The post-thaw motility of crane sperm was improved (P<0.05) with a combination of 18.0%, 24.0% or 26.0% DMA and 30mM ATP. Moreover, in the presence of osmoprotectants, crane sperm motility decreased as the osmoprotectant concentration increased. The lowest concentration of ATP also improved crane sperm viability post-thaw, especially for DMA concentrations 18% or greater. The combination of sucrose and trehalose improved (P<0.05) crane sperm viability only with 6% and 10% DMA. These data affirm that there are avian-specific differences in sperm survival after cryopreservation and suggest that post-thaw survival can be enhanced by including species-based osmoprotectant/ATP combinations in a cryodiluent where DMA is the cryoprotectant.

Seropositivity and Risk Factors Associated with Toxoplasma gondii Infection in Wild Birds from Spain

Toxoplasma gondii is a zoonotic intracellular protozoan parasite of worldwide distribution that infects many species of warm-blooded animals, including birds. To date, there is scant information about the seropositivity of T. gondii and the risk factors associated with T. gondii infection in wild bird populations. In the present study, T. gondii infection was evaluated on sera obtained from 1079 wild birds belonging to 56 species (including Falconiformes (n = 610), Strigiformes (n = 260), Ciconiiformes (n = 156), Gruiformes (n = 21), and other orders (n = 32), from different areas of Spain. Antibodies to T. gondii (modified agglutination test, MAT titer ≥1∶25) were found in 282 (26.1%, IC95%:23.5–28.7) of the 1079 birds. This study constitute the first extensive survey in wild birds species in Spain and reports for the first time T. gondii antibodies in the griffon vulture (Gyps fulvus), short-toed snake-eagle (Circaetus gallicus), Bonellis eagle (Aquila fasciata), golden eagle (Aquila chrysaetos), bearded vulture (Gypaetus barbatus), osprey (Pandion haliaetus), Montagus harrier (Circus pygargus), Western marsh-harrier (Circus aeruginosus), peregrine falcon (Falco peregrinus), long-eared owl (Asio otus), common scops owl (Otus scops), Eurasian spoonbill (Platalea leucorodia), white stork (Ciconia ciconia), grey heron (Ardea cinerea), common moorhen (Gallinula chloropus); in the International Union for Conservation of Nature (IUCN) “vulnerable” Spanish imperial eagle (Aquila adalberti), lesser kestrel (Falco naumanni) and great bustard (Otis tarda); and in the IUCN “near threatened” red kite (Milvus milvus). The highest seropositivity by species was observed in the Eurasian eagle owl (Bubo bubo) (68.1%, 98 of 144). The main risk factors associated with T. gondii seropositivity in wild birds were age and diet, with the highest exposure in older animals and in carnivorous wild birds. The results showed that T. gondii infection is widespread and can be at a high level in many wild birds in Spain, most likely related to their feeding behaviour.

Comparative cryopreservation of avian spermatozoa: effects of freezing and thawing rates on turkey and sandhill crane sperm cryosurvival.

A comparative approach was used to evaluate semen cooling rates, thawing rates and freezing volume on the cryosurvival of avian sperm. Turkey (Meleagris gallopavo) and sandhill crane (Grus canadensis) sperm were cryopreserved with dimethylacetamide (DMA) concentrations ranging from 6% to 26%. Experiments evaluated the efficacy of (1) rapid, moderate and slow cooling rates, (2) rapid and slow thawing rates, and (3) final volume of semen frozen (0.2 mL compared to 0.5 mL). For crane sperm only, additional experiments were conducted to evaluate the effect of sucrose on cryosurvival. The functionality of frozen/thawed crane sperm was evaluated by fertility trials. For all studies, sperm viability was assessed using the nigrosin-eosin stain. Higher percentages of crane and turkey sperm maintained intact membranes when frozen with moderate or slow cooling rates compared to rapid cooling rates (P<0.05), regardless of DMA concentration. Turkey sperm viability was not affected by thawing rate at any DMA concentration (P>0.05). Crane sperm viability was only affected by thawing rate for the 24% DMA treatment, where moderate thawing was better than slow thawing (P<0.05). Sperm viability was not affected by the semen volume used for freezing for either species (P>0.05). The percentage of membrane-intact crane sperm at lower DMA concentrations was improved by addition of 0.1M sucrose (P<0.05) but not 0.29 M NaCl. The mean fertility rate from frozen/thawed crane semen was 57.5%, and 71.4% of the fertile eggs hatched. The viability of crane sperm was always greater than turkey sperm, regardless of cooling rate, thawing rate or volume of semen frozen. These data verify avian-specific differences in sperm cryosurvival, further emphasize the need for species specific studies to optimize cryopreservation protocols.

Producing progeny from endangered birds of prey: treatment of urine-contaminated semen and a novel intramagnal insemination approach.

Abstract Wild raptors brought into an ex situ environment often have poor semen quality that is further compromised by urine contamination. Generally, it is believed that in birds, artificial insemination into the cloaca or caudal vagina of females requires large doses of high-quality spermatozoa to maximize fertility. In an effort to define and overcome some of the challenges associated with reproduction in wild raptors, the objectives of this study were to 1) evaluate the frequency, impact, and remediation of urine contamination in fresh ejaculates for the purpose of maintaining sperm motility and viability in vitro, and 2) develop a deep insemination method that allows low numbers of washed sperm to be placed directly into the magnum to increase the probability of producing fertilized eggs. The species evaluated include golden eagle (Aquila chrysoetos), imperial eagle (A. adalberti), Bonellis eagle (Hiernaetus fasciatus), and peregrine falcon (Falco peregrinus). Semen samples were collected and pooled by species, and a minimum of 25 pooled ejaculates per species were evaluated for urine contamination, pH, sperm viability, and sperm motility; the samples were either unwashed or washed in neutral (pH 7.0) or alkaline (pH 8.0) modified Lakes diluent. Female golden eagles and peregrine falcons were inseminated via transjunctional, intramagnal insemination with washed spermatozoa from urine-contaminated samples. Urine contamination occurred in 36.8 ± 12.8% (mean ± SEM) golden eagle, 43.1 ± 9.1% imperial eagle, 28.7 ± 16.1% Bonellis eagle, and 48.2 ± 17.3% peregrine falcon ejaculates. The pH in urine-contaminated semen samples ranged from 6.48 ± 0.3 to 6.86 ± 0.2, and in noncontaminated samples it ranged from from 7.17 ± 0.1 to 7.56 ± 0.1. Sperm viability and motility were reduced (P < 0.05) in all species for unwashed vs. washed sperm after 30 min incubation at room temperature. Two peregrine falcon chicks and one golden eagle chick hatched after intramagnal insemination. This study demonstrates that urine contamination, a common and lethal acidifier in manually collected raptor ejaculates, can be circumvented by immediate, gentle seminal washing. Furthermore, these processed sperm, when deposited by transjunctional intramagnal insemination, can produce live young.

Serologic Testing for Avian Influenza Viruses in Wild Birds: Comparison of Two Commercial Competition Enzyme-Linked Immunosorbent Assays

Abstract Serologic testing of wild birds for avian influenza virus (AIV) surveillance poses problems due to species differences and nonspecific inhibitors that may be present in sera of wild birds. Recently available competitive enzyme-linked immunosorbent assay (cELISA) kits offer a new species-independent approach. In this study we compare two commercial competitive cELISAs, using a total of 184 serum and plasma samples from 23 species of wild birds belonging to 10 orders. Thirteen samples were from experimentally high pathogenicity AI and low pathogenicity AI infected red-legged partridges (Alectoris rufa), 77 samples were from a flock of sentinel hybrid ducks confirmed infected by AI by real-time PCR, and 94 samples were from wild birds admitted to a rehabilitation center. Both ELISAs detected AI antibodies in the experimentally infected partridges, whereas hemagglutination inhibition (HI) was negative. Concordance in results between the two ELISAs was 51.5%. When specific subtype-H5/H7 HI-positive samples were considered for comparison, ELISA 1 appeared to perform better on ducks, whereas ELISA 2 appeared to perform better in other wild bird species. Overall, 68.2% of H5/H7 positive samples tested positive by ELISA 1 and 36% by ELISA 2. Both ELISAs detected AIV-antibody–positive samples negative by specific HI against 9 of the 16 existing hemagglutinin (HA) subtypes. Presumably this reflects either higher sensitivity of cELISA when compared to HI, presence of antibodies against HA subtypes not tested, or unspecific reactions. Performance of ELISA 1 on ducks appears to be comparable to in-house cELISA previously used by other authors in wild birds, but requires a relatively large sample volume. Alternatively, although ELISA 2 required a smaller sample volume, it was less effective at identifying HI-positive samples. The results reflect the necessity of validation of cELISA tests for individual species or at least families, as required by the OIE.

THU0259 Frequency and pattern of the uveitis in spondyloarthritis with biological therapy

Background Uveitis is the most frequent extra-articular manifestation (EAM) of spondyloarthritis (SpA). Its prevalence is approximately 30% and increases with the duration of the SpA. The characteristic pattern is anterior, acute, recurrent and unilateral uveitis. However, the frequency and characteristics of uveitis in SpA treated with biological therapy (BT) are unknown. Objectives The main target is to describe the frequency and characteristics of uveitis in SpA with BT in a single centre. Methods Descriptive and retrospective study (January 2003-December 2017) of SpA that develops uveitis in a reference hospital. The epidemiological variables, type of SpA, presence of uveitis and its characteristics, presence of BT at the time of onset and treatment received are collected. For the analysis, frequencies and percentages were used in qualitative variables, and mean and standard deviation (SD) for quantitative variables. Statistical analysis was performed with IBM SPSS v.23. Results We studied 246 patients with SpA. The subtypes of SpA were: ankylosing spondylitis (AS) (n=125, 50.8%), psoriatic arthritis (PsA) (n=101, 41.1%), undifferentiated SpA (n=13, 5.3%), non-radiographic axial Spa (n=3, 1.2%), enteropathic arthropathy (n=3, 1.2%) and reactive arthritis (n=1, 0.4%). Uveitis was observed in 41 patients (16.7%) after an average time of development of 109.47 (73.9) months of the SpA. The incidence rate was 5.5 cases of uveitis/100 patients-year of follow-up. 70.7% were men and the mean age(SD) was 47.4 (12.06) years. 87.8% of the cases were HLAB27 positive and had a family history of SpA 41.5%. Uveitis was observed in 33 patients (80.5%) with AS, in 6 (14.6%) with PsA, in 1 (2.4%) with non-Rx axial SpA and in 1 (2.4%) with undifferentiated SpA. (table 1) The uveitis pattern was anterior (100%), acute (92.7%), unilateral (87.8%) and in 12.2% bilateral (80% in PsA). At the time of onset of uveitis, the mean ESR was 30.11 mm1ªh, CRP 3.56 mg/dL, DAS28 3.66 and BASDAI 3.21. Regarding the diagnosis of SpA, uveitis was after (85.4%), before (12.2%) and simultaneous (2.4%). At the time of the onset of uveitis, 14 patients (34.1%) were with BT (35.7% etanercept, 28.6% infliximab, 21.4% adalimumab, 7.1% golimumab and 7.1% certolizumab). BT was modified in 3 of the cases. The treatment of uveitis was topical (78%), corticoids in oral regimen (57.5%), conventional DMARDs (12.5%), with methotrexate predominating in 60% of cases and BT (15%). The most used biologics were adalimumab (50%), infliximab (33.3%) and sekukinumab (16.7%).Abstract THU0259 – Table 1 Characteristics of the UVEITIS in SpA subtypes Conclusions In our series, uveitis was observed in 16.7% of patients with SpA of which 80.5% were AS and 14.6% PsA. The most frequent uveitis was anterior, unilateral, acute and recurrent. In PsA, the association with HLA B27 was less frequent and was more bilateral. In most cases, the diagnosis was later than the SpA. Disclosure of Interest None declared

AB1373 Urinary protein profile comparison between sle patients with and without renal involvement

Background Lupus nephropathy (NL) is an important cause of morbidity and mortality in patients with Systemic Lupus Erythematosus (SLE). The objective of the renal biopsy is to determine the type of glomerulonephritis that the patient presents to direct treatment. Considering that it is a specialised technique and not risk free, a proteomics study is proposed to determine biomarkers that help us to differentiate patients diagnosed with SLE with and without renal involvement. Objectives To determine if there is a different pattern of proteins between patients diagnosed with SLE with and without renal involvement. Methods We selected 12 patients diagnosed with SLE with renal involvement and 14 patients diagnosed with SLE without renal involvement. There were no differences between groups according to race, gener and age. The patients were classified as high, low or negative level of proteinuria in the urine. A 24 hour urine sample was obtained for analysis. Results We have done a Principal Component Analysis (PCA) where we can see differences between samples from patients who have high level of proteinuria in 24 hours and patients who have not renal involvement. Patients with positive proteinuria but not high level are a little confuse figure 1. A total of 292 proteins (identified with at least two peptides with a FDR<1%) were quantified and further considered in the analysis. The Student’s T-test analysis reflected the differential presence of 147 proteins (p<0.01). Of these, 130 were less abundant in the urine of the patients with renal damage, whereas 17 showed the opposite pattern, being more abundant in the patients with affected renal function. Consistent with the nature of the sample, the Gene Ontology (GO analysis) of the whole list of identified proteins revealed the presence of extracellular (277 proteins, p=2.25E-171) and secretion-related proteins (49 proteins, p=1.1E-09), among others. Proteins related to defensive processes were prominent among them. Interestingly, the subset of proteins whose abundance increases upon renal damage is comprised of typical highly-abundant serum proteins. These proteins render a large number of peptides, suggesting they are very abundant. This protein pattern may reflect the higher albuminuria characteristic of patients with affected renal function. On the other hand, a number of proteins became significantly less abundant upon renal damage. The presence of highly abundant serum proteins in the urine of patients with compromised renal function may explain this phenomenon, since this will provoke a dramatic reduction in the relative abundance of the proteins already present in their urine.Abstract AB1373 – Figure 1 Conclusions A different protein pattern is observed between the two groups of patients, so in a more detailed study we can indicate if some of these can serve as prognostic markers for this type of patients. Disclosure of Interest None declared