Experimental infection of wild-caught European rabbits (Oryctolagus cuniculus) with Sarcoptes scabiei from a naturally infected wild rabbit.

Scabies was recently reported for the first time in the European wild rabbit, Oryctolagus cuniculus (Lagomorpha: Leporidae). We experimentally exposed 10 seronegative wild-caught rabbits to skin from a mangy wild rabbit. Serological, physiological, parasitological and histopathological changes were recorded. Three rabbits developed antibodies at 2-5 weeks post-infection (w.p.i.), two of which then developed lesions at 7 w.p.i. One of these had a small area of alopecia on the hind limb that healed naturally within 1 week; the other developed more extensive lesions restricted to the hind limbs (as typically observed in wild rabbits) that lasted until the rabbit died (12.5 w.p.i.). The third rabbit died of trauma 5 w.p.i. before developing any lesions. Antibodies in the healed rabbit disappeared from serum at 8 w.p.i., whereas antibody levels in the sick rabbit increased until its death. Disseminated intravascular coagulation and hepatic necrosis, probably arising from a concomitant infection with rabbit haemorrhagic disease virus, were the likely final cause of death in this rabbit. The mangy rabbit that served as a donor died of a multifocal fibrinosuppurative pneumonia that may have been secondary to the skin bacterial pyoderma.

Cell counts and survival to vitrification of bovine in vitro produced blastocysts subjected to sublethal high hydrostatic pressure.

This work analyses the effects of a high hydrostatic pressure (HHP) treatment on in vitro survival of in vitro produced (IVP) bovine embryos vitrified with the Cryologic Vitrification Method (CVM). Consequences on embryo quality in terms of cell proliferation and differentiation, and levels of embryonic Heat Shock Protein 70 (Hsp-70) were also examined. Day 7 and 8 bovine in vitro-produced blastocysts were submitted to an HHP treatment (60 MPa, at 32 °C for 1 h) and allowed to recover for 1 or 2 h in culture medium. The HHP treatment did not improve blastocyst survival rates after vitrification/warming. Survival (24 h post-warming) and hatching (48 h post-warming) rates were 79.3 ± 4.9 and 51.8 ± 4.2 vs 73.9 ± 4.2 and 44.7 ± 4.1 for untreated controls and HHP-treated embryos, respectively. Total cell numbers measured in fresh embryos were reduced after 1 h at 32 °C, with or without HHP treatment, indicating that cell proliferation was stopped as a result of stress. Vitrified HHP-treated embryos that hatched at 48 h after warming showed increased cell numbers in their ICM compared with untreated controls (50.2 ± 3.1 vs 38.8 ± 2.7), indicating higher embryo quality. Treatment of blastocysts with HHP did not alter the level of the Hsp-70 protein. In our conditions, HHP treatment did not affect the cryoresistance of these embryos. However, combination of HHP treatment and vitrification in fibreplugs resulted in an increase in the ICM cell number of hatched embryos 48 h post-warming.

Mechanical and tribological characterization of TiB2 thin films.

Titanium Diboride (TiB2) presents high mechanical and physical properties. Some wear studies were also carried out in order to evaluate its tribological properties. One of the most popular wear tests for thin films is the ball-cratering configuration. This work was focused on the study of the tribological properties of TiB2 thin films using micro-abrasion tests and following the BS EN 1071-6: 2007 standard. Due to high hardness usually patented by these films, diamond was selected as abrasive on micro-abrasion tests. Micro-abrasion wear tests were performed under five different durations, using the same normal load, speed rotation and ball. Films were deposited by unbalanced magnetron sputtering Physical Vapour Deposition (PVD) technique using TiB2 targets. TiB2 films were characterized using different methods as Scanning Electron Microscopy (SEM), Energy Dispersive X-ray Spectroscopy (EDS), Atomic Force Microscopy (AFM), X-ray Diffraction (XRD), Electron Probe Micro-Analyser (EPMA), Ultra Micro Hardness and Scratch-test Analysis, allowing to confirm that TiB2 presents adequate mechanical and physical properties. Ratio between hardness (coating and abrasive particles), wear resistance and wear coefficient were studied, showing that TiB2 films shows excellent properties for tribological applications.

Female segregation patterns of the putative Y-chromosome-specific microsatellite markers INRA124 and INRA126 do not support their use for cattle population studies.

Here we tested the segregation and paternal compatibility of markers INRA124 and INRA126 on female DNA in 10 different cattle families, in order to clarify the usefulness of these microsatellites for the study of male-mediated population processes in cattle. Their performance was compared with that of four microsatellites located in the PAR-BTAY (UMN0108, UMN0803, UMN0929 and UMN0905) and another one male-specific microsatellite (INRA189). INRA124 and INRA126 amplified the same sized fragment in both sexes. Same size alleles were sequenced and the high homology found allowed us to rule out non-specific female amplification. INRA124 showed full parental compatibility, whilst the locus INRA126 showed 55% parental incompatibility. Based on these observations, it is recommended that markers INRA124 and INRA126 should not be used in studies to characterize male-mediated genetic events in cattle.

Pathology, isolation and molecular characterisation of a ranavirus from the common midwife toad Alytes obstetricans on the Iberian Peninsula.

We describe the pathology, isolation and characterisation of a virus responsible for an outbreak of a systemic haemorrhagic disease causing high mortality in tadpoles of the common midwife toad Alytes obstetricans in the 'Picos de Europa' National Park in northern Spain. The virus, provisionally designated as the common midwife toad virus (CMTV), was isolated from homogenates of visceral tissue from diseased toad tadpoles following inoculation on epithelioma papulosum cyprini (EPC) cells. Molecular characterisation of the virus, including sequence analysis of the DNA polymerase and major capsid protein genes, showed that the isolated virus was a ranavirus with marked sequence identity to other members of the genus Ranavirus. A rabbit antiserum raised against purified virions was prepared and used to definitively demonstrate systemic distribution of the virus in diseased tadpoles, indicating that the isolated virus was the primary pathogen.

Sarcoptic mange in red deer from Spain: improved surveillance or disease emergence?

Concern about emerging diseases has risen in recent years, and multihost situations have become increasingly relevant for wildlife management and conservation. We present data on Asturias, northern Spain, where 80 mangy red deer (Cervus elaphus) have been found since the beginning of the epizootic in chamois (Rupicapra pyrenaica parva) in 1993. We combine field and necropsy data with the results of a serosurvey using an in-house ELISA test to evaluate if deer mange due to Sarcoptes scabiei is an emerging disease in this area. The mean number of deer mange cases per year was 5, with a maximum of 16. No significant relationship was detected between monthly temperatures, rainfall or number of days with snow cover and the annual number of sarcoptic mange cases in red deer. Only 4 mangy red deer (5%) were detected outside the limits of scabietic chamois distribution during the same year, and all were less than 2500 m away from that limit. The longest distance reported between two consecutive mangy deer locations was 18 km. Mange cases were significantly more frequent in stags than in hinds and in adults than in juvenile deer. The time of the first mange detection in chamois in each sector, year with minimum number of chamois recorded, year with maximum chamois population decline rate and chamois density offered no significant correlation with red deer mange cases appearance moment and frequency. In the mange affected area, ELISA testing of 327 blood samples from hunter-harvested deer without obvious mange-compatible lesions revealed only 4 seropositive animals. All 83 sera from hunting preserves without clinical cases yielded negative ELISA results. According to these epidemiological data mange does not seem to threaten red deer populations in Asturias. However, continued monitoring of deer health and ELISA testing for sarcoptic mange is advisable.

Clinical course and pathogenicity of variant rabbit haemorrhagic disease virus in experimentally infected adult and kit rabbits: Significance towards control and spread.

RHDVb has become the dominant RHDV on the Iberian Peninsula. A better understanding of its pathogenicity is required to aid control measures. Thus, the clinical course, humoral immune response, viraemia and kinetics of RHDV-N11 (a Spanish RHDVb isolate) infection in different tissues at both viral RNA and protein levels were studied in experimentally infected young and adult rabbits. The case fatality rate differed between the two age groups, with 21% of kits succumbing while no deaths were observed in adults. Fever and viremia were strongly associated with death, which occurred 48 h post infection (PI) too fast for an effective humoral immune response to be mounted. A significant effect on the number of viral RNA copies with regard to the variables age, tissue and time PI (p < 0.0001 in all cases) was detected. Histological lesions in infected rabbits were consistently more frequent and severe in liver and spleen and additionally intestine in kits, these tissues containing the highest levels of viral RNA and protein. Although no adults showed lesions or virus antigen in intestine, both kits and adults maintained steady viral RNA levels from days 1 to 7 PI in this organ. Analysis revealed the fecal route as the main dissemination route of RHDV-N11. Subclinically infected rabbits had detectable viral RNA in their faeces for up to seven days and thus may play an important role spreading the virus. This study allows a better understanding of the transmission of this virus and improvement of the control strategies for this disease.

ELISA for detection of variant rabbit haemorrhagic disease virus RHDV2 antigen in liver extracts.

The emergence and rapid spread of variant of the rabbit hemorrhagic disease virus (RHDV2) require new diagnostic tools to ensure that efficient control measures are adopted. In the present study, a specific sandwich enzyme-linked immunosorbent assay (ELISA) for detection of RHDV2 antigens in rabbit liver homogenates, based on the use of an RHDV2-specific monoclonal antibody (Mab) 2D9 for antigen capture and an anti-RHDV2 goat polyclonal antibody (Pab), was developed. This ELISA was able to successfully detect RHDV2 and RHDV2 recombinant virions with high sensitivity (100%) and specificity (97.22%). No cross-reactions were detected with RHDV G1 viruses while low cross-reactivity was detected with one of the RHDVa samples analyzed. The ELISA afforded good repeatability and had high analytical sensitivity as it was able to detect a dilution 1:163,640 (6.10ng/mL) of purified RHDV-N11 VLPs, which contained approximately 3.4×108molecules/mL particles. The reliable discrimination between closely related viruses is crucial to understand the epidemiology and the interaction of co-existing pathogens. In the work described here we design and validate an ELISA for laboratory based, specific, sensitive and reliable detection of RHDVb/RHDV2. This ELISA is a valuable, specific virological tool for monitoring virus circulation, which will permit a better control of this disease.

Lambs are Susceptible to Experimental Challenge with Spanish Goat Encephalitis Virus.

Spanish goat encephalitis virus (SGEV) is a member of the genus Flavivirus, family Flaviviridae, and causes encephalomyelitis in goats. The aim of this study was to determine whether sheep are susceptible to experimental challenge with SGEV by two different routes. The results show that SGEV can infect sheep by both the subcutaneous and intravenous routes, resulting in neurological clinical disease with extensive and severe histological lesions in the central nervous system. Lambs challenged subcutaneously developed more severe lesions on the ipsilateral side of the brain, but the lesion morphology was similar irrespective of the route of challenge. The clinical presentation, pathogenesis, lesion morphology and distribution shows that SGEV is very similar to louping ill virus (LIV) and therefore any disease control plan must take into account any host species and SGEV vectors as potential reservoirs. Furthermore, discriminatory diagnostics need to be applied to any sheep or goat suspected of disease due to any flavivirus in areas where SGEV and LIV co-exist.

Vaccination against Louping Ill Virus Protects Goats from Experimental Challenge with Spanish Goat Encephalitis Virus.

Spanish goat encephalitis virus (SGEV) is a recently described member of the genus Flavivirus belonging to the tick-borne encephalitis group of viruses, and is closely related to louping ill virus (LIV). Naturally acquired disease in goats results in severe, acute encephalitis and 100% mortality. Eighteen goats were challenged subcutaneously with SGEV; nine were vaccinated previously against LIV and nine were not. None of the vaccinated goats showed any clinical signs of disease or histological lesions, but all of the non-vaccinated goats developed pyrexia and 5/9 developed neurological clinical signs, primarily tremors in the neck and ataxia. All non-vaccinated animals developed histological lesions restricted to the central nervous system and consistent with a lymphocytic meningomyeloencephalitis. Vaccinated goats had significantly (P <0.003) greater concentrations of serum IgG and lower levels of IgM (P <0.0001) compared with unvaccinated animals. SGEV RNA levels were below detectable limits in the vaccinated goats throughout the experiment, but increased rapidly and were significantly (P <0.0001) greater 2-10 days post challenge in the non-vaccinated group. In conclusion, vaccination of goats against LIV confers highly effective protection against SGEV; this is probably mediated by IgG and prevents an increase in viral RNA load in serum such that vaccinated animals would not be an effective reservoir of the virus.

Concomitance and interactions of pathogens in the Iberian wolf (Canis lupus).

With the aim of improving our understanding of their epidemiological features, exposure to or presence of Canine Parvovirus (CPV), Canine Distemper Virus (CDV), Leishmania infantum and Sarcoptes scabiei were studied in 88 wild wolves from Asturias (Northern Spain) by means of long-term (2004-2010) serological and molecular data. Individual and population factors and the possible interactions between them were also statistically analyzed for better understanding the contact/presence of studied pathogens. The overall seroprevalence values were 19%, 61%, 20% and 0% for CDV, CPV, S. scabiei and Leishmania, respectively, while a 46% of studied wolves showed Leishmania genetic material presence. Sarcoptic mange, CDV and CPV showed higher seroprevalence values in the areas with higher wolf densities, and a positive association between CDV and S. scabiei antibody responses was detected. Reported data highlight the need of considering concomitant pathogens and their possible interactions for a better understanding of diseases and their management in wildlife.

Identification and expression of a Fasciola hepatica gene encoding a gut antigen protein bearing repetitive sequences.

A Fasciola hepatica cDNA clone of about 2 kb was isolated from an expression library by immunological screening using blood serum from an experimentally infected calf. The cDNA clone hybridised to a RNA of about 3 kb in a Northern blot experiment. The nucleotide sequence of the cDNA revealed the presence of an open reading frame of 1636 bp which encoded 24 tandemly arranged 20-amino acid-long repeats, followed by 65 non-repeated residues preceding the stop codon. This antigen was expressed in Escherichia coli as beta-galactosidase fusion proteins which were used for the production of specific antibodies. Immunofluorescence studies using specific antifusion sera revealed that the antigen was specifically expressed in the parasite intestine epithelial cells. Due to its early appearance it might be possible to design diagnostic assays based on this repeated antigen for identification of recently infected animals.

Cloning and expression in Escherichia coli of a Fasciola hepatica gene encoding a calcium-binding protein.

A Fasciola hepatica cDNA clone of 994 bp was isolated from an adult worm cDNA expression library using a rabbit serum against the excretory-secretory antigens. The nucleotide sequence of the cDNA clone revealed the presence of an open reading frame of 572 bp which encoded a 22 kDa polypeptide (Fh22) showing putative EF-hand domains. This gene was expressed in Escherichia coli and the recombinant protein used for the production of specific antibodies. Immunoblotting studies using the anti-Fh22 serum showed the presence of a polypeptide of similar molecular mass in the excretory-secretory extract of the adult parasite. The recombinant Fh22 polypeptide showed calcium-dependent electrophoretic mobility (decreased with Ca2(+)-ions and increased with EGTA). The observed behaviour of recombinant Fh22 in gel filtration experiments also suggested calcium-induced conformational changes.

The amino terminal sequence of VP60 from rabbit hemorrhagic disease virus supports its putative subgenomic origin.

Direct determination of the amino acid sequence of VP60 from rabbit hemorrhagic disease virus is impeded by the presence of a blocked N-terminus. Chemical cleavage of VP60 using cyanogen bromide allowed the identification and purification of two oligopeptides showing identical amino acid composition, one of which had its amino terminus blocked. Automated sequential degradation of the unblocked CNBr- peptide yielded the amino acid sequence EGKARTAPQGEAA. This sequence is identical to the deduced amino acid sequence following the first AUG codon found at position +10 at the 5'-end of the 2.4 kb subgenomic mRNA. These data favor the hypothesis that this viral polypeptide is mainly produced from the subgenomic mRNA and not from the genomic RNA by processing of the putative polyprotein generated from the major open reading frame.

In vitro translation of a subgenomic mRNA from purified virions of the Spanish field isolate AST/89 of rabbit hemorrhagic disease virus (RHDV).

Purified preparations of the Spanish field isolate of rabbit hemorrhagic disease virus AST/89 were found to contain the plus-stranded genomic RNA of more than 7.4 kilobases (kb) and large amounts of a subgenomic mRNA of 2.4 kb. The smaller RNA was translated in vitro and shown to code for a 60 kDa protein which was immunoprecipitated using anti-RHDV as well as anti-VP60 sera.

Immunogenic properties of rabbit haemorrhagic disease virus structural protein VP60 expressed by a recombinant baculovirus: an efficient vaccine.

We have constructed a recombinant baculovirus containing the gene encoding the structural protein VP60 from the Spanish field isolate AST/89 of rabbit haemorrhagic disease virus (RHDV). Infection of cultured Spodoptera frugiperda Sf9 cells with this recombinant virus resulted in the production of high yields of VP60 protein which did not seem to assemble to form virus like particles, but was antigenically similar to the corresponding viral protein obtained from purified virions. A VP60-dose study showed that the recombinant protein was able to elicit a protective response in rabbits against a nasal challenge with 100 LD50 of RHDV. The effective dose able to protect 50% of the animals in the absence of adjuvant was found to be 10-25 micrograms of recombinant VP60.

Variable performance of a human derived Sarcoptes scabiei recombinant antigen ELISA in swine mange diagnosis.

The performance of an indirect ELISA test based on Sarcoptes scabiei var hominis recombinant antigen Ssλ20ΔB3 (rec-ELISA), to diagnose pig mange was investigated in 15 experimentally infected and non-infected pigs and 692 commercial pigs from 16 herds in southeast Spain. These latter animals included 6-7 month old fatteners (13 herds), 11-12 month old replacement sows (1 herd) and ≥24 month old breeding sows (7 herds). All pigs were examined for mites in ear skin scrapings and the presence of S. scabiei-associated macroscopic dermatitis; moreover, fatteners were also tested for antibodies against porcine viruses including: Aujeszky disease virus (ADV), swine influenza virus (SIV), type 2 porcine circovirus (PCV2) and porcine respiratory and reproductive syndrome virus (PRRSV). S. scabiei and chronic hyperkeratotic dermatitis were detected in breeding sows from 6 herds. Mite prevalence in other pigs was 83% in replacement sows, 0% in 7 fattener's herds and 3-82% in other fattener's herds. All fattener herds had pigs with acute hypersensitivity dermatitis and the percentage of affected pigs and lesion area was significantly greater in S. scabiei infected ones. Rec-ELISA relative optical densities (RODs) were greater in older than in young pigs, as well as in infected compared to non-infected pigs. However, RODs differed significantly between infected individuals, regardless of age and origin (commercial or experimental) and the herd prevalence of S. scabiei. Low repeatability between ELISA microtiter plates, suggesting variable specific antibody binding to antigen, are likely partly responsible for ROD variation. Other potential causes of variation were examined in fatteners using random effects logistic regression analysis, after defining a seropositivity threshold value with receiver-operating characteristics (ROC) analysis. The logistic model indicated that seropositivity was associated with large dermatitis areas and with the only herd with low PCV2 seroprevalence. Pigs with more extensive dermatitis may have older infections and more rec-ELISA detectable antibodies. The possibility that PCV2, a recognized immunosupressor, depresses antibody production against S. scabiei infection merits further attention. In summary, results indicate some potential of the studied rec-ELISA as a complementary tool for herd-level swine mange diagnosis, and that work to reduce internal and external sources of assay variation is essential.

On the aggregated nature of chronic Sarcoptes scabiei infection in adult pigs.

The prevalence and body distribution of Sarcoptes scabiei and associated dermatitis was investigated in sows and boars from four herds with long standing mange. Macroscopic hyperkeratotic dermatitis (crusted mange) was present in 1-6% of herd sows. Mite estimated prevalence (95% CI) in ear scrapings was 11% (6-17%) including 100% (13/13) and 2% (3/134) in sows with and without crusted mange, respectively, and the later had very few mites compared to the former. S. scabiei body distribution and dermatitis were further investigated in 59-64 skin scrapings/sow taken post-mortem from four culled sows including two (sows 1 and 2) with and two (sows 3 and 4) without crusted mange. The proportion of skin samples with eggs, instars or adults was 59% in sow 1, 84% in sow 2, 0% in sow 3 and 3% in sow 4. S. scabiei distribution in sows 1 and 2 ranged from being present in all skin ear and head samples to absent in those from the inner side of the limbs and mammary glands. Crusted lesions were observed in the skin of the ears, neck and lower limbs and contained the largest mite populations. Histopathological analysis of skin samples identified mites, inflammatory cellular infiltrate (mainly lymphocytes, neutrophils and eosinophils) and hyperkeratosis, acanthosis and spongiosis in 78%, 54%, 20% and 25% of samples from sows 1, 2, 3 and 4, respectively, being lesion severity positively associated to mite presence. The study provides further evidence that in herds with long-standing exposure to S. scabiei, infection becomes highly overdispersed with large mite populations present only in a few pigs and in specific body areas. Although the reasons for mite aggregation have not been identified, it is important controlwise because treating or eliminating a few and easy to identify heavily infected adult pigs, should markedly decrease the herd's parasite load and reduce the use of acaridal drugs.

Widespread exposure to Sarcoptes scabiei in wild European rabbits (Oryctolagus cuniculus) in Spain.

Sarcoptic mange was recently described in the wild European rabbit (Oryctolagus cuniculus) in north-eastern Mediterranean Spain, the first such infection reported in this species anywhere in the world. This finding has created concern in conservationists and game managers given that an outbreak of mange after a translocation would have catastrophic consequences for naïve rabbit populations in other parts of Spain. A retrospective serosurvey using an 'in house' ELISA test based on the use of a recombinant antigen aimed at determining the rates of contact with Sarcoptes scabiei was carried out on sera from 966 rabbits collected between 1993 and 2010 in Spain. Antibodies were found in 13% of wild rabbits in 60% of the 53 areas surveyed, as well as in 16 of the 17 Spanish provinces and islands studied. Seropositive rabbits were found amongst the oldest samples analyzed and in all studied years. Antibodies were also detected in 36% of rabbits from the protected island of Dragonera, where rabbits have probably not been released since the 1970s. On Mallorca, where 89 rabbits were inspected for both lesions and antibodies, the prevalence of lesions (5.6%) was much lower than the seroprevalence (22.5%), indicating that rabbits often survive infection or that ELISA detects infected rabbits before they develop visible lesions. Seroprevalence was higher in areas with medium levels of rabbit abundance, no restocking and high rainfall. The results show that mange is widespread in rabbits and that the mite is not a recent introduction. Thus, sarcoptic mange could be considered as an enzootic disease in the wild rabbit and so prophylactic measures implemented during rabbit translocations are to be encouraged to avoid local outbreaks in naïve populations.

Outbreak of common midwife toad virus in alpine newts (Mesotriton alpestris cyreni) and common midwife toads (Alytes obstetricans) in northern Spain: a comparative pathological study of an emerging ranavirus.

This report describes the isolation and characterisation of the common midwife toad virus (CMTV) from juvenile alpine newts (Mesotriton alpestris cyreni) and common midwife toad (CMT) tadpoles (Alytes obstetricans) in the Picos de Europa National Park in Northern Spain in August 2008. A comparative pathological and immunohistochemical study was carried out using anti-CMTV polyclonal serum. In the kidneys, glomeruli had the most severe histological lesions in CMT tadpoles, while both glomeruli and renal tubular epithelial cells exhibited foci of necrosis in juvenile alpine newts. Viral antigens were detected by immunohistochemical labelling mainly in the kidneys of CMT tadpoles and in ganglia of juvenile alpine newts. This is the first report of ranavirus infection in the alpine newt, the second known species to be affected by CMTV in the past 2 years.