Monitoring of the novel rabbit haemorrhagic disease virus type 2 (GI.2) epidemic in European wild rabbits (Oryctolagus cuniculus) in southern Spain, 2013-2017.

Rabbit hemorrhagic disease (RHD) is a highly infectious disease in European rabbits (Oryctolagus cuniculus), caused by a virus belonging to the genus Lagovirus (RHDV; family Caliciviridae). In 2010, a new genotype of RHDV (RHDV2 or RHDVb, currently designated GI.2) emerged in France, affecting both domestic rabbits, even those vaccinated for the classical RHDV genotypes (currently designated GI.1) and wild rabbits. GI.2 was subsequently identified in other European countries. The aim of the present study was to monitor the GI.2 epidemic in wild rabbits in Andalusia (southern Spain) during the period 2013-2017. At the beginning of summer 2013, high mortalities were detected in wild rabbit populations in southern Spain. A total of 96 affected hunting or protected areas were surveyed. The first outbreak was observed on June 2013. The number of outbreaks sharply increased in 2013 and 2014, with a decreasing trend being observed during the following years. The spatial distribution of GI.2 was not homogeneous, since most of the detected outbreaks were concentrated in the western part of Andalusia. The outbreaks peaked in winter and spring and have been detected in the last five consecutive years, which suggests endemic circulation of GI.2 in wild rabbit populations in Spain. A total of 190 dead rabbits from 87 of the 96 areas surveyed were collected during the study period. Mortality affected rabbits of different age classes, including kittens. RT-PCR confirmed the presence of GI.2 RNA in the livers of 185 of the 190 (97.4%) rabbits. Phylogenetic analysis performed on eleven samples collected in different provinces of Andalusia between 2013 and 2017, showed high nucleotide identity with GI.2 strains Spain, France and Portugal. The results constitute an important step in understanding of the emergence and spread of GI.2 in this country and will provide valuable information for the development of surveillance programs in Europe.

Monitoring of the Bagaza virus epidemic in wild bird species in Spain, 2010.

By the end of August 2010, high mortalities in red-legged partridges (Alectoris rufa) and pheasants (Phasianus colchicus) were detected in several hunting states in the province of Cádiz (southern Spain). Retrospective epidemiological studies revealed that the first clinical signs had been observed in late July-early August. The most common clinical signs were incoordination, disorientation and ataxia. The estimated mean morbidity rates were 37% in partridges and 11% in pheasants. The estimated mean mortality rates were 23% in partridges and 6% in pheasants. The estimated mean case-fatality rates were 38% and 8% in partridges and pheasants, respectively. A total of 19 clinically affected birds from 18 affected hunting states were analysed between August and November 2011. Histopathological analyses revealed encephalitis, myocarditis, leiomyositis, meningoencephalitis and neuritis as the most frequently observed lesions. Molecular analyses identified Bagaza virus (BAGV) as the causative agent of the epidemic. Further studies are needed to determine the source of introduction of the virus into Europe and to elucidate whether wild birds play a role in the epidemiology of BAGV. Surveillance in susceptible bird species, including partridges and pheasants, may be useful for the early detection of BAGV in an area.

Role of testosterone and photoperiod on seasonal changes in horn growth and sperm variables in the Iberian ibex: a model for polygynous wild bovids.

This work examines the effect of testosterone secretion and photoperiod on seasonal changes in horn growth and sperm variables in the Iberian ibex (Capra pyrenaica), here used as a model for polygynous wild bovids. The hypothesis that high levels of testosterone provide an endocrine signal that inhibits horn growth in autumn was tested by assessing the effect of cyproterone acetate (CA), an anti-androgen, administered in October - coinciding with the period of natural increases in plasma testosterone concentrations - under different photoperiodic conditions (natural photoperiod and artificial long days). The persistence of horn growth during autumn in all ibexes held under the long-day photoperiodic conditions clearly shows that horn growth regulation in the mating season is primarily modulated by day length and not by a fall in testosterone concentration. A retrospectively designed second experiment involving testosterone propionate (TP) administration in April (when horns are growing) was then undertaken to confirm that high levels of testosterone do not inhibit horn growth. Overall, the results strongly suggest that the rise in testosterone secretion during the autumn mating season does not act as an endocrine signal for the arrest of horn growth, although the rate of horn growth before the mating season may be related to springtime testosterone levels. A direct relationship was seen between the rate of horn growth and the incidence of sperm abnormalities. Neither CA treatment in October nor TP administration in April affected the studied sperm variables. By contrast, CA treatment plus artificial long days in autumn had a negative effect on sperm motility and sperm morphology.

Prevalence of Toxoplasma gondii and Neospora caninum antibodies in Spanish ibex (Capra pyrenaica hispanica).

A cross-sectional study was carried out on Spanish ibex populations in Southern Spain to assess the seroprevalence of Toxoplasma gondii and Neospora caninum and to investigate the risk factors associated with these infections. Using the modified agglutination test, the seroprevalence to T. gondii was 27.5% (146/531; CI(95%), 23.7-31.3), and this seropositivity significantly increased with age. Among adults, statistically significant differences were observed between geographical locations and over different sampling years. Thirty of 531 (5.6%) ibex had antibodies to N. caninum using a competitive ELISA, of which 27/30 (5.1%; CI(95%), 3.1-7.1) were confirmed as seropositive by the indirect fluorescent antibody test. This study is the first to report the presence of N. caninum antibodies in Spanish ibex and also indicates widespread exposure of this species to T. gondii. The findings indicate that ibex are more exposed to T. gondii than to N. caninum in their natural environment and there is little evidence of co-infection with both parasites. The seroprevalence levels reported suggest a role for ibex in the sylvatic cycle of both parasites with potentially important environmental and public health implications.

Effects of anesthetic protocols on electroejaculation variables of Iberian ibex (Capra pyrenaica).

The effects of three intramuscular anesthesia protocols--detomidine 190 µg/kg plus ketamine 2 mg/kg, detomidine 270 µg/kg plus ketamine 1.4 mg/kg, and tiletamine 3.4 mg/kg plus zolazepam 3.4 mg/kg--on penis protrusion and ejaculation variables were compared in nine captive Spanish ibex (Capra pyrenaica) subjected to electroejaculation. Body temperature, heart, and respiratory rates, as well as a number of plasma biochemical variables were also recorded prior to and during anesthesia. The detomidine plus ketamine protocols induced bradycardia and increased respiratory rate. However, the tiletamine/zolazepam protocol did not affect heart and respiratory rates. None of the three protocols caused a substantial change in rectal temperature, yet all protocols caused a significant increase in plasma glucose levels. Differences in anesthetic protocols did not affect sperm quality or quantity. However, choice of anesthetic protocol affected (P<0.05) the degree of penis protrusion and the electrical pulse sequence required to achieve ejaculation. Results of this study support a recommendation of detomidine 270 µg/kg plus ketamine 1.4 mg/kg for anesthesia of Spanish ibex undergoing electroejaculation.

Cryopreservation of Spanish ibex (Capra pyrenaica) sperm obtained by electroejaculation outside the rutting season.

The effectiveness of electroejaculation for obtaining Spanish ibex sperm samples for freeze preserving outside the rutting season was evaluated-the aim being to optimise biological resources for the establishment of germplasm banks. The effect of different egg yolk concentrations (6% or 12%, v/v) in diluents of different buffer composition (Tris-citric acid buffer or Tes-Tris buffer) on frozen-thawed samples of the above also investigated. Experiments were undertaken with six ibex males in February-May, and involved four different semen samples from each animal with four combination of extender, respectively: Tes-Tris-glucose (TTG)-6% egg yolk, TTG-12% egg yolk, Tris-citric acid-glucose (TCG)-12% egg yolk, TCG-6% egg yolk. The results show that electroejaculation is a useful way of obtaining sperm samples from Spanish ibex outside the rutting season (i.e., at a time coinciding with plasma testosterone levels of <0.4ng/ml). According to the results of the eosin-nigrosin staining and the hypo-osmotic swelling test, the freezing-thawing process significantly reduced the viability and membrane integrity of the spermatozoa extended with TTG-6% egg yolk, TTG-12% egg yolk, and TCG-12% egg yolk, but did not affect these variables in spermatozoa extended with TCG-6% egg yolk. Therefore, the use of Tris-citric acid-based extenders containing low concentrations of egg yolk is recommended for cryopreserving Spanish ibex spermatozoa obtained by electroejaculation outside the rutting season.