Pentobarbital intoxication as a potential underlying cause for electrocution in a bearded vulture.

A bearded vulture (Gypaetus barbatus) found dead in northern Spain presented external lesions consistent with electrocution as the cause of death. During forensic examination, macroscopic lesions suggested potential comorbidity, so samples were collected for molecular and toxicological analyses. Gastric content and liver were analysed for toxic substances, and pentobarbital (a common pharmaceutical used for euthanasia in domestic animals) was detected at a concentration of 37.3 and 0.05 µg/g, respectively. Other toxicological, viral and endoparasite analyses (avian malaria, avian influenza and flaviviruses) were negative. Thus, although the cause of death was electrocution, pentobarbital intoxication likely impaired the equilibrium and reflexes of the individual, possibly causing the bird to contact energized wires that it would not have otherwise. These results underline the importance of comprehensive analysis of forensic cases of wildlife deaths and reveal barbiturate poisoning as an additional threat for the conservation of the bearded vulture in Europe.

High prevalence and intensity of Stephanurus dentatus in a population of wild boar (Sus scrofa) in south western Spain.

In the period from October 2016 to February 2017, the urinary tracts of 390 wild boar (Sus scrofa) from four areas of south central Spain (102 from Doñana National Park; 150 from Sierra Morena and the Toledo Mountains; 84 from Sierra Nevada; 54 from Sierra de Cazorla, Segura y Las Villas Natural Park) were examined for the presence of adult specimens of Stephanurus dentatus (Nematoda: Strongyloidea). This parasite was only detected in the wild boar population of Doñana National Park, with high prevalence (76.5±4.2%; 78/102), mean intensity (43.2±4.4) and mean abundance (33.1±3.8). Juvenile wild boar had significantly lower prevalence and abundance than subadult and adult wild boar. The intensity of infestation was significantly higher in male than in female wild boar. The detection of a focus of S. dentatus infestation in the wild boar population in Doñana National Park will provide further opportunities for understanding the epidemiology of this parasite.

Experimental North American West Nile Virus Infection in the Red-legged Partridge (Alectoris rufa).

After the introduction of West Nile virus (WNV) into North America, bird mortalities associated with West Nile disease have dramatically increased in this continent and, to a lesser extent, in Europe. The different West Nile disease incidence in birds in these 2 continents demands an explanation, and experimental studies can provide important information. The authors inoculated thirteen 9-week-old red-legged partridges (Alectoris rufa) with 10(7)plaque-forming units of a WNV strain isolated in New York in 1999. The objective was to study the pathogenesis of the infection in a native Euro-Mediterranean bird species with a WNV strain known to be highly pathogenic for numerous native American bird species. Additionally, the authors evaluated the dynamics of inflammatory cell activation and recruitment into the brain. WNV was detected in tissues 3 days postinoculation (dpi), and the birds developed macroscopic and microscopic lesions. Two partridges succumbed to the disease. The most affected tissues were the heart, brain, and spinal cord. The main microscopic findings were the presence of mononuclear infiltrates in the heart and brain, gliosis, and degeneration and necrosis of cardiomyocytes and neurons. These lesions were aggravated in the birds that died or were euthanized 7 dpi or later. In the brain, there was an upregulation of microglial cells and astrocytes and an increase in the number of T cells, especially after 7 dpi. These results show that this WNV strain is of moderate virulence for the red-legged partridge and that WNV-infected red-legged partridges develop an immune cell response in the brain similar to that of mammals.

Oculopathologic findings in flavivirus-infected gallinaceous birds.

Using eye samples of nine 9-week-old experimentally West Nile virus (WNV)-infected red-legged partridges (Alectoris rufa), time course of lesions and WNV antigen appearance in ocular structures were examined. In addition, eye samples of 6 red-legged partridges and 3 common pheasants (Phasianus colchicus) naturally infected with Bagaza virus (BAGV) were used to study lesions and flavivirus antigen distribution in relation to apparent blindness in the former. The rapid onset of microscopic lesions and early presence of viral antigen in the eye of experimentally WNV-infected partridges, prior to the central nervous system involvement, suggested hematogenous spread of the virus into the eye. BAGV-infected partridges had a more pronunced inflammatory reaction and more widespread flavivirus antigen distribution in the retina compared with pheasants and experimentally fatally WNV-infected partridges. Our results suggest that flavivirus replication and development of lesions in ocular structures of gallinaceous game birds vary with the specific virus and host species involved.

Protection of red-legged partridges (Alectoris rufa) against West Nile virus (WNV) infection after immunization with WNV recombinant envelope protein E (rE).

West Nile virus (WNV) is maintained in nature in an enzootic transmission cycle between birds and mosquitoes, although it occasionally infects other vertebrates, including humans, in which it may result fatal. To date, no licensed vaccines against WNV infection are available for birds, but its availability would certainly benefit certain populations, as birds grown for restocking, hunting activities, or alimentary purposes, and those confined to wildlife reservations and recreation installations. We have tested the protective capability of WNV envelope recombinant (rE) protein in red-legged partridges (Alectoris rufa). Birds (n=28) were intramuscularly immunized three times at 2-weeks interval with rE and a control group (n=29) was sham-immunized. Except for 5 sham-immunized birds that were not infected and housed as contact controls, partridges were subcutaneously challenged with WNV. Oropharyngeal and cloacal swabs and feather pulps were collected at several days after infection and blood samples were taken during vaccination and after infection. All rE-vaccinated partridges elicited anti-WNV antibodies before challenge and survived to the infection, while 33.3% of the sham-immunized birds succumbed, as did 25% of the contact animals. Most (84%) unvaccinated birds showed viremia 3 d.p.i., but virus was only detected in 14% of the rE vaccinated birds. WNV-RNA was detected in feathers and swabs from sham-immunized partridges from 3 to 7 d.p.i., mainly in birds that succumbed to the infection, but not in rE vaccinated birds. Thus, rE vaccination fully protected partridges against WND and reduced the risk of virus spread.

A colibacillosis outbreak in farmed red-legged partridges (Alectoris rufa).

Colibacillosis in different forms is responsible for significant economic losses in the poultry industry worldwide. Escherichia coli strains frequently implicated in poultry disease are designated as avian pathogenic E. coli (APEC). Natural infections and disease due to APEC have been described in wild birds, but not as yet in red-legged partridges. During an outbreak in an experimental partridge farm, 23 of 43 1-day-old chicks belonging to the same batch died. Putative APEC strains were detected and isolated both in cloacal swabs and in tissues originating from the same individuals and from different birds showing similar clinical signs. This is the first study that identifies APEC strains linked to a colibacillosis outbreak in farmed red-legged partridges, and also confirms the importance of farmed partridges as fecal carriers and potential spreaders of APEC.

Factors affecting red deer skin test responsiveness to bovine and avian tuberculin and to phytohaemagglutinin.

We studied the effect of management on the responsiveness of red deer (Cervus elaphus) to skin testing with mycobacterial and non-mycobacterial antigens. We hypothesized that individuals from populations of the same species under different management conditions would have a different immune responsiveness. Deer sampled in this study included 1041 adult animals from 6 Spanish farms and 111 adult wild deer. We injected four sites of the neck with 0.1 ml bovine purified protein derivative (PPD), 0.1 ml avian PPD, 0.1 ml negative control PBS and 0.1 ml of Phytohaemagglutinin (PHA, containing 250 microg) as positive control, and measured the skin fold increase at time 72 h. Bovine PPD reactors were identified in 5 of 6 farms and among wild deer. Apparent prevalence among wild deer (18.9%) was not significantly higher than among farmed deer (14.5%). Avian PPD reactors were found among all 7 study populations, but apparent prevalence was lower among wild deer (<1%) than among farmed deer (12.6%; p<0.001). Deer management (farmed versus wild) was identified as a key factor affecting deer skin fold thickness increase in response both to mycobacterial (bPPD and aPPD) and non-mycobacterial antigens (PHA). The differences occurred in the same sense, regardless of some interactions; farmed deer showing higher values. The PHA skin fold increase was not affected by the PPD skin test results. We propose that using PHA as a positive control may help in the interpretation of between-population differences in tuberculin responses.

Implementing artificial insemination as an effective tool for ex situ conservation of endangered avian species.

Approximately 503 of the known species of birds are classified as 'endangered' or 'critical'. Captive propagation programs have proven useful in maintaining genetic diversity and restoring wild populations of certain species, including the Peregrine falcon, California condor and Whooping crane. Artificial insemination (AI) has the potential of solving problems inherent to reproductive management of small, closed populations of endangered birds, including dealing with demographic instability, physical and behavioral disabilities, sexual incompatibility, lack of synchrony, and need to maintain gene diversity. In this review, we address the necessary methods and factors that allow AI to be applied effectively to manage rare bird populations. It is clear that semen availability and quality are the greatest limiting factors to implementing consistently successful AI for birds. Behavioral sensitivity to animal handling and the ability to minimize stress in individual birds also are keys to success. Multiple, deep vaginal inseminations can improve fertility, particularly when semen quality is marginal. Laparoscopic methods of semen transfer also have produced fertile eggs. All of these practices leading to successful AI remain dependent on having adequate basic knowledge on female reproductive status, copulatory behavior, endocrine profiles and duration of fertility, especially as related to oviposition. The overall greatest challenge and highest priority is defining these normative traits, which are highly species-specific.

The effects of sex and age on phytohaemagglutinin skin-testing of deer.

AIM: To determine if there are sex- or age-related differences in the increase in skinfold thickness in response to the mitogen phytohaemagglutinin (PHA) in red deer. METHODS: One dose of 250 mug PHA was injected intradermally in the right side of the neck, and phosphate buffered saline (PBS) was injected at a second site as a control, in 110 (51 males and 59 females) captive Iberian red deer (Cervus elaphus hispanicus), ranging in age from 21 months to > or =5 years. Skinfold thicknesses were measured immediately before and 72 h following injection. RESULTS: There was a significant effect of gender on the average increase in skinfold thickness; males had greater increases (8.8 (SEM 0.57) mm) than females (4.23 (SEM 0.39) mm) after correcting for other confounding variables. No age-related differences were evident, but differences between sexes were more marked with increasing age. CONCLUSIONS AND CLINICAL RELEVANCE: Effects of gender, probably due to differences in energetic and reproductive constraints in red deer, should be taken into account when interpreting skinfold-test data, both in ecology and in the control of tuberculosis (Tb). Males tend to have a thicker skin than females, so skinfold increase relative to the thickness of the skin, rather than skinfold increase per se, should be used as a more appropriate measure of skinfold increase. This may also have clinical relevance in the interpretation of tuberculin skin testing.

Estimation of European wild boar relative abundance and aggregation: a novel method in epidemiological risk assessment.

Wild boars are important disease reservoirs. It is well known that abundance estimates are needed in wildlife epidemiology, but the expense and effort required to obtain them is prohibitive. We evaluated a simple method based on the frequency of faecal droppings found on transects (FBII), and developed a spatial aggregation index, based on the runs test statistic. Estimates were compared with hunting data, and with porcine circovirus and Aujeszky's disease virus seroprevalences and Mycobacterium tuberculosis complex and Metastrongylus spp. prevalence. The FBII and the aggregation index were correlated with the hunting index, but both of the former estimates correlated better than the latter with the disease prevalences. Hence, at least in habitats with high wild boar densities, the FBII combined with the aggregation index constitutes a cheap and reliable alternative for wild boar abundance estimation that can be used for epidemiological risk assessment, even outside the hunting season and in areas with no available data on hunting activities.

Optimal dose and timing in phytohaemagglutinin skin-testing of deer.

AIM: To establish the optimal dose of the mitogen phytohaemagglutinin (PHA) and the optimal time for measuring increased skin-fold thickness in red deer following intradermal injection, as an indicator of cell-mediated immune response. METHODS: Three doses (10, 50 and 250 microg) of PHA were injected intradermally in the right side of the neck, and phosphate buffered saline (PBS) was injected at a fourth site as a control, in 20 captive Iberian red deer (Cervus elaphus hispanicus) hinds. Skin-fold thicknesses were measured at 0, 12, 24, 36, 48, 60, 72, 84 and 96 h following injection. RESULTS: The highest dose of PHA tested (250 microg) resulted in a clear and long-lasting cellular response; increases in skin-fold thickness between 48 and 84 h post-injection varied minimally and response correlated positively with liveweight. No correlations with liveweight and no clear increases in skin-fold thickness occurred at the lower doses of PHA or the PBS. CONCLUSIONS AND CLINICAL RELEVANCE: This technique could be applied with minimal training and without specialised equipment in deer, for immunological and ecological research.

Changes in parasite transmission stage excretion after pheasant release.

The production of parasite transmission stages was investigated in the faeces of 77 farm-bred ring-necked pheasants (Phasianus colchicus). Coccidian oocysts (Eimeria sp.), and nematode eggs (Heterakis sp., and Capillaria-like eggs) were recovered before and after release but all birds were treated prior to release. Treatment with fenbendazole significantly reduced the abundance of transmission-stage excretion for all parasites, and reduced the prevalence in the case of Eimeria sp. and Heterakis sp. Nonetheless, a significant increase in the excretion abundance for all parasites and in the prevalence of Eimeria sp. and Heterakis sp. was found after release. Eggs of Ascaridia sp. were found only after releasing, suggesting infection ocurred in the wild. A negative relationship was found between the pheasant body condition and Heterakis excretion abundance and a higher abundance of Capillaria sp. eggs in female birds. No significant relationship was found between parasite excretion abundance and pheasant survival. Despite this, results suggest that an increase in the excretion of parasite transmission stages follows the release of captive pheasants into the wild. This can in part explain restocking failures, but also means that autochtonous free-living birds may become exposed to new and potentially harmful pathogens. To avoid these risks it is proposed that improved prophylactic measures should be taken.

Avoiding bias in parasite excretion estimates: the effect of sampling time and type of faeces.

The study of host-parasite relationships usually requires reliable estimates of parasite intensity, which is often estimated from parasite propagule concentration in faeces. However, parasite excretion in faeces may be subject to variation due to endogenous or exogenous factors that must be identified to obtain reliable results. We analysed the effect of the hour of sample collection on propagule counts of 2 intestinal parasites infecting the red-legged partridge: the capillarid nematode Aonchoteca caudinflata and coccidia of the genus Eimeria (Protozoa). Also, we test whether there are differences in propagule counts between caecal and intestinal faeces. Individual faecal samples from infected birds were collected daily at 4 different hours during several days. The hour of the day exerted a very strong effect on propagule counts, excretion of both types of parasites showing a clear and constant increase from dawn to dusk. Also, capillarid eggs were more abundant in intestinal than in caecal faeces, whereas the inverse pattern was found for coccidian oocysts. Standardization of the hour of sample collection or statistical control of this variable is recommendable to prevent bias. Similarly, in bird species with long caeca, consistent collection of one type of faeces may avoid significant errors in parasite burden estimates.

Serosurvey of Aujeszky's disease virus infection in European wild boar in Spain.

Serum samples from 693 hunted wild boar (Sus scrofa) were analysed by means of a blocking ELISA technique, and the mean (se) prevalence of antibodies to Aujeszky's disease virus was 44 (4) per cent. All the seropositive wild boar were from south central Spain, except for one from central Spain, close to the main positive area. In this area, where large game species are increasingly managed for hunting, the seroprevalence was affected by the type of management. More intensively managed populations had a higher prevalence than wild boar living in natural situations, and the seroprevalence increased with the age of the animals; the seroprevalence was higher in females in all age groups. The seroprevalence in males more than one year old peaked after the breeding season, whereas females of the same age had a higher and constant seroprevalence throughout the year.

The risks of translocating wildlife. Pathogenic infection with Theileria sp. and Elaeophora elaphi in an imported red deer.

It is well known that the translocation of wild animals poses risks of the introduction of pathogens into populations, and regulations and recommendations regarding quarantine and screening protocols for wild animals do exist. Less is known about the infection of imported animals with local endemic pathogens. A red deer stag that had been imported from Germany was found recumbent and died from hemolytic anaemia and a process of exertional myopathy. Infection with Theileria sp. was detected in thin blood smears and confirmed by PCR and sequencing. In addition, massive parasitation by Elaeophora elaphi, a parasite endemic to Iberian red deer, was detected. Sequence comparison between the 18S rRNA gene sequence determined that the Theileria strain involved in this case had a 99.7% identity with a Theileria sp. strain obtained from sika-deer, and 95.3% identity with T. cervi. Using sequence distance analysis, the strain from red deer grouped with isolates from Cervus spp. as opposed to isolates from Odocoileus spp. and bovines. Both detected parasites are of little pathogenicity to local red deer, but were pathogenic for the imported red deer from Northern Europe. This case demonstrates that local endemic pathogens may pose naive translocated animals at risk, and illustrates the need for thorough examination and planification of translocation protocols.

Descriptive study of an avian pox outbreak in wild red-legged partridges (Alectoris rufa) in Spain.

This study describes the dynamics and epidemiology of an outbreak of avian pox in free-living, red-legged partridges (Alectoris rufa) in southern Spain. Between March 2000 and January 2001, 115 free-living, red-legged partridges (70 juveniles, 45 adults) were captured and radio-tagged. This, together with the necropsy of 44 carcasses (10 juveniles, 34 adults) found in the study area, and the inspection of 108 shot birds (74 juveniles, 34 adults) after a hunting drive in October, permitted a close monitoring of the course of the disease. Forty-one per cent of radio-tagged juveniles but none of 45 radio-tagged adults showed pox-like lesions at the time of capture, recapture, or necropsy. At least 40% of the juveniles that survived into the hunting season, but only 2.9% of the adults inspected at the same time, showed lesions suggestive of infection with avian poxvirus. The survival of juveniles during the peak of the outbreak was much lower than that of the adults, but we found no significant differences between the survival probabilities of juvenile partridges with and without pox-like lesions. Nevertheless, some birds may have developed lesions after their capture. The occurrence and course of the disease in a managed area with intense predator control underlines the need for studies on the combined influence of diseases and predators on population dynamics. Also the need for early detection of diseases for the management of game species is emphasized.

Bilateral ovarian teratoma in a free-living Iberian red deer (Cervus elaphus hispanicus).

CASE HISTORY: A 8.5 x 5.7 x 5 cm irregularly shaped mass was detected in the location of the left ovary of a 9-year-old Iberian red deer (Cervus elaphus hispanicus) hind shot during a hunting drive. PATHOLOGICAL FINDINGS: On the cut surface, the mass consisted of numerous fluid- or keratin-filled inter-communicating cysts, separated by connective tissue containing solid nodules of dark-coloured foci and hairy skin. The right ovary was of normal size, but contained a 1.5 cm mass of hairy skin and spongy structures. Histologically, the large mass comprised differentiated structures such as cartilage, fibrous connective tissue, smooth muscle, stratified squamous keratinised epithelium, cuboidal multi-layered and respiratory epithelium, clusters of pigment-laden cells, hair follicles, nervous tissue and glandular structures. The hind had a grossly and microscopically normal uterus that contained a 449.5 g female fetus. DIAGNOSIS: Bilateral mature ovarian teratomas. CLINICAL RELEVANCE: This case describes the first known description of an ovarian teratoma in a free-living red deer. The tumour had unique features, such as bilateral presence and concurrency with a normal pregnancy.

Chaunocephalus ferox in free-living white storks in central Spain.

Ten out of 42 (23.8%) white storks (Ciconia ciconia) admitted to two rehabilitation centers in central Spain had lesions caused by the trematode Chaunocephalus ferox in the small intestinal wall. Fourteen of the examined birds were adults, five were subadults, and 23 were chicks of various ages. Parasitation was 32% (n = 8) in chicks and 13% (n = 2) in adult birds, whereas no juvenile bird was affected. Among dead birds, stork chicks affected by C. ferox lesions had a lower body weight (2196.1 g, SD = 814.2) than storks without lesions (2965.8 g, SD = 742.9, P < 0.05). Two chicks were additionally infected with Salmonella subspecies I serotype enteritidis 1,9,12: g, m:1, 7. Prevalence of the parasite in the examined birds was lower than in a population of Asian open-billed storks (Anastomus oscitans), in which it was pathogenic due to the destruction of the tunica muscularis and formation of large granulomatous lesions in the wall of the postduodenal portion of the small intestine. Pathogenic alterations caused by C. ferox are presumed to be related to numbers of adults present. Because storks admitted to rehabilitation centers suffer stress due to various reasons that may lower their immune response and exacerbate existing infections, the analysis of fecal sediments of white storks admitted for rehabilitation is recommended.

Pathology of avian pox in wild red-legged partridges (Alectoris rufa) in Spain.

The diagnosis and pathology of an avian pox outbreak in free-living red-legged partridges in Cádiz, Southern Spain, is described. Diagnosis of the disease was based on histopathology, ultrastructural examination of, and virus isolation from lesions of necropsied animals. Lesions were present mainly in juvenile partridges (41%), and were observed primarily on the dorsal part of the digits or on the hock joint. The lesions ranged from small wartlike nodules to large tumor-like lesions. The presence of acute lesions of any grade as opposed to absence of lesions or healed lesions adversely affected body condition of the partridges (P <.01). Further investigations on the epidemiology of the disease and on the relation of the isolated strains to other avian poxviruses are under way.