Ovis Aries Musimon Research Articles

Identification of Ancestry Informative Marker (AIM) Panels to Assess Hybridisation between Feral and Domestic Sheep.

Simple SummaryOnce present in the entirety of Europe, mouflon (wild sheep) became extinct due to intense hunting, but remnant populations survived and became feral on the Mediterranean islands of Corsica and Sardinia. Although now protected by regional laws, Sardinian mouflon is threatened by crossbreeding with domestic sheep causing genetic hybridisation. The spread of domestic genes can be detrimental for wild populations as it dilutes the genetic features that characterise them. This work aimed to identify diagnostic tools that could be applied to monitor the level of hybridisation between mouflon and domestic sheep. Tens of thousands of genetic markers known as single nucleotide polymorphisms (SNPs) were screened and we identified the smallest number of SNPs necessary to discriminate between pure mouflon and sheep. We produced four SNP panels of different sizes which were able to assess the hybridisation level of a mouflon and we verified that the SNP panels efficacy is independent of the domestic sheep breed involved in the hybrid. The implementation of these results into actual diagnostic tools will help the conservation of this unique and irreplaceable mouflon population, and the methodology applied can easily be transferred to other case studies of interest. Hybridisation of wild populations with their domestic counterparts can lead to the loss of wildtype genetic integrity, outbreeding depression, and loss of adaptive features. The Mediterranean island of Sardinia hosts one of the last extant autochthonous European mouflon (Ovis aries musimon) populations. Although conservation policies, including reintroduction plans, have been enforced to preserve Sardinian mouflon, crossbreeding with domestic sheep has been documented. We identified panels of single nucleotide polymorphisms (SNPs) that could act as ancestry informative markers able to assess admixture in feral x domestic sheep hybrids. The medium-density SNP array genotyping data of Sardinian mouflon and domestic sheep (O. aries aries) showing pure ancestry were used as references. We applied a two-step selection algorithm to this data consisting of preselection via Principal Component Analysis followed by a supervised machine learning classification method based on random forest to develop SNP panels of various sizes. We generated ancestry informative marker (AIM) panels and tested their ability to assess admixture in mouflon x domestic sheep hybrids both in simulated and real populations of known ancestry proportions. All the AIM panels recorded high correlations with the ancestry proportion computed using the full medium-density SNP array. The AIM panels proposed here may be used by conservation practitioners as diagnostic tools to exclude hybrids from reintroduction plans and improve conservation strategies for mouflon populations.

Exposure to Toxoplasma gondii in zoo animals in Spain

A serosurvey was performed to assess exposure to Toxoplasma gondii in zoo animals in Spain and to determine the dynamics of seropositivity in some zoo species over time. Sera from 393 zoo animals belonging to 91 species were collected in eight zoos in Spain between 2007 and 2019. Additionally, 39 of the 393 animals from five of the analyzed zoos were longitudinally sampled during the same study period. Antibodies to T. gondii were detected in 165 (42.0 %; CI95 %: 37.1–46.9) of 393 animals by the modified agglutination test (MAT, cut-off = 25). Antibody titers of 25, 50, 100 and ≥500 were found in 25 (15.2 %), 65 (39.4 %), 26 (15.8 %), and 49 (29.7 %) of the seropositive animals, respectively. Feeding habits (carnivorous species) was a risk factor (OR = 5.6; P < 0.001; CI95 %: 3.8–9.0) potentially associated with T. gondii exposure. Eighteen (46.2 %) of the 39 animals longitudinally sampled were always seropositive and five animals (12.8 %) (two spotted hyaena [Crocuta crocuta], one blesbok [Damaliscus pygargus], one white rhinoceros [Ceratotherium simum] and one mouflon [Ovis aries musimon]) seroconverted during the study period. Our results evince a wide circulation of T. gondii in zoos in Spain, including wild felids. Zoo animals can be useful sentinel species to monitor T. gondii circulation in epidemiological scenarios with a close human-wildlife interface. Control measures should be implemented in zoo parks to minimize the risk of exposure of zoo species to T. gondii.

Contrasting patterns of sexually selected traits in Mediterranean and continental populations of European mouflon.

The expression of sexually selected traits in highly dimorphic ungulates may be influenced by environmental quality. Variations in habitat conditions can impose different constraints on the allocation of energy resources to male life‐history traits, and possibly alter the female preferences for specific features. Here, we compared the horn growth patterns in male European mouflon Ovis aries musimon living in different habitats (Mediterranean vs. continental) but sharing a common genetic origin. We hypothesized that the expression of sexually selected traits such as horn development should be promoted in more favorable habitat conditions (i.e., Mediterranean). Using linear mixed models on data retrieved from individuals harvested under the same hunting regime, we found longer horns and greater individual variance in horn segment length in the Mediterranean population than in the continental one. Furthermore, Mediterranean rams showed no evidence of compensatory horn growth, as opposed to the continental rams. Unexpectedly, horn base circumference was greater in the continental habitat than in the Mediterranean one. The overall results suggest different patterns of investment in horns in the two populations, with seemingly stronger pressure and consequences of sexual selection on mouflon rams living in more favorable environments. Although the role of hunters' selectivity cannot be excluded a priori, our data suggest that the differences in the expression of sexually selected traits in our study populations may be influenced by environmental conditions. Because sexual selection can impose substantial fitness costs on individuals, further investigations on the trade‐offs between reproduction and survival would improve our understanding of the dynamics of mouflon populations living in different environmental conditions.

Deer keds on wild ungulates in northern Italy, with a taxonomic key for the identification of Lipoptena spp. of Europe.

Deer keds (Lipoptena spp.) are blood-sucking ectoparasites of domestic and wild animals, and also accidentally of humans. In Europe, five Lipoptena spp. have been recorded, although the lack of specific taxonomic keys has often led to mistaken identification or to missing data. The present study aimed to develop an identification key of the European species and also to identify Lipoptena spp. found on wild ungulates in northern Italy. In total, 390 hippoboscids were collected from Rupicapra rupicapra, Capreolus capreolus, Cervus elaphus and Ovis aries musimon in an Alpine area of Italy. After morphological identification, 140 specimens were subjected to phylogenetic analysis based on mitochondrial (CO1) and nuclear (CAD) gene sequences. Despite the expected presence of slight morphological variations, all specimens examined were identified both microscopically and molecularly as Lipoptena cervi (100% identity for both CO1 and CAD genes). The massive increase in wild ungulate populations can favour the possibility of detecting other species of Lipoptena. The identification keys proposed in the present study may help with monitoring the presence of Lipoptena species, particularly in European countries where this ectoparasite is neglected and for which various data (from diffusion to control methods) are still missing.

Density-dependent decline of early horn growth in European mouflon

The development of horns in Caprinae can be largely influenced by food-limiting factors such as population density and climate, as well as by negative evolutionary responses to size-selective harvesting. In this study, we investigated the effects of population density, environmental covariates and trophy hunting on horn development in a population of European mouflon Ovis aries musimon introduced to the Mediterranean region of Croatia in the early 1980s. The study population was subject to commercial trophy hunting on males since the mid-1980s. This allowed to analyse the temporal trend in early horn growth in 341 rams legally culled. Cohort-based linear model and analysis of deviance (ANODEV) revealed a significant negative trend in early horn growth, with a decline of ca. 10% over only 15 cohorts (1993–2007). The increase in population density (by ca. 600%) and summer temperature, selected via LASSO regression, explained about 61% and 13% of the decline in early horn growth, as revealed by the ANODEV R2. Our results suggest a prominent role of food-limiting factors in the decline of weapon size. We also found a negative relationship between age at death and early horn growth in our study population, which suggests the occurrence of hunters’ selectivity towards large horns. The effect of trophy hunting on the decline in horn size, however, is difficult to quantify. Our analysis was largely influenced by the rapid increase of mouflon density after introduction, thereby limiting the possibility to detect potential effects of hunting selection, although the large number of rams shot before 5 years of age may possibly lead to undesirable consequences of trophy hunting on mating success. To clarify the consequences of commercial trophy hunting on the mouflon population, the long-term pattern of horn growth and the age-dependent male siring success should be further investigated.

Long-term dynamics of Mycoplasma conjunctivae at the wildlife-livestock interface in the Pyrenees.

Functional roles of domestic and wild host populations in infectious keratoconjunctivitis (IKC) epidemiology have been extensively discussed claiming a domestic reservoir for the more susceptible wild hosts, however, based on limited data. With the aim to better assess IKC epidemiology in complex host-pathogen alpine systems, the long-term infectious dynamics and molecular epidemiology of Mycoplasma conjunctivae was investigated in all host populations from six study areas in the Pyrenees and one in the Cantabrian Mountains (Northern Spain). Detection of M. conjunctivae was performed by qPCR on 3600 eye swabs collected during seven years from hunted wild ungulates and sympatric domestic sheep (n = 1800 animals), and cluster analyses of the strains were performed including previous reported local strains. Mycoplasma conjunctivae was consistently detected in three Pyrenean chamois (Rupicapra p. pyrenaica) populations, as well as in sheep flocks (17.0% of sheep) and occasionally in mouflon (Ovis aries musimon) from the Pyrenees (22.2% in one year/area); statistically associated with ocular clinical signs only in chamois. Chamois populations showed different infection dynamics with low but steady prevalence (4.9%) and significant yearly fluctuations (0.0%– 40.0%). Persistence of specific M. conjunctivae strain clusters in wild host populations is demonstrated for six and nine years. Cross-species transmission between chamois and sheep and chamois and mouflon were also sporadically evidenced. Overall, independent M. conjunctivae sylvatic and domestic cycles occurred at the wildlife-livestock interface in the alpine ecosystems from the Pyrenees with sheep and chamois as the key host species for each cycle, and mouflon as a spill-over host. Host population characteristics and M. conjunctivae strains resulted in different epidemiological scenarios in chamois, ranging from the fading out of the mycoplasma to the epidemic and endemic long-term persistence. These findings highlight the capacity of M. conjunctivae to establish diverse interactions and persist in host populations, also with different transmission conditions.

First record of Gongylonema nepalensis in domestic and wild ruminants in Europe

The gullet worm, Gongylonema pulchrum, is cosmopolitan in distribution, infecting a variety of mammals including domestic and wild ruminants. Gongylonema nepalensis recently collected from the esophageal epithelium of water buffaloes in Nepal was separated from G. pulchrum based on its distinctly shorter left spicule relative to body length and unique nucleotide sequences of the ribosomal RNA gene (rDNA) and the mitochondrial cytochrome c oxidase subunit 1 gene (cox-1). During meat inspections at four abattoirs on Sardinia Island, Italy, 25 Gongylonema worms were collected from one each individual of cattle (n=8), sheep (n=7), goats (n=4), and mouflon (Ovis aries musimon; n=6), and characterized morphologically and genetically. Intriguingly, all of the collected worms from these ruminants were G. nepalensis, exhibiting comparable body lengths to G. pulchrum in cattle from other regions but with significantly shorter left spicules like G. nepalensis (less than 20.9% of the entire body length in contrast to 21.8–65.6%, the reported proportion of G. pulchrum). Furthermore, the rDNA nucleotide sequences of these worms from different ruminant species on Sardinia Island were almost identical to each other and to Nepalese G. nepalensis isolates. With the exception of one worm from a sheep (displaying a single nucleotide substitution), the 369-bp cox-1 nucleotide sequences of all the Sardinian G. nepalensis isolates from the different host sources were absolutely identical, forming a clade with Nepalese G. nepalensis isolates and not G. pulchrum isolates. The present study reveals that G. nepalensis is not a local parasite in the Indian subcontinent (Nepal), but instead has a certain geographical distribution in Europe and takes several ruminant species as a definitive host.

Genomic signatures of adaptive introgression from European mouflon into domestic sheep

Mouflon (Ovis aries musimon) became extinct from mainland Europe after the Neolithic, but remnant populations from the Mediterranean islands of Corsica and Sardinia have been used for reintroductions across Europe since the 19th-century. Mouflon x sheep hybrids are larger-bodied than mouflon, potentially showing increased male reproductive success, but little is known about genomic levels of admixture, or about the adaptive significance of introgression between resident mouflon and local sheep breeds. Here we analysed Ovine medium-density SNP array genotypes of 92 mouflon from six geographic regions, along with data from 330 individuals of 16 domestic sheep breeds. We found lower levels of genetic diversity in mouflon than in domestic sheep, consistent with past bottlenecks in mouflon. Introgression signals were bidirectional and affected most mouflon and sheep populations, being strongest in one Sardinian mouflon population. Developing and using a novel approach to identify chromosomal regions with consistent introgression signals, we infer adaptive introgression from mouflon to domestic sheep related to immunity mechanisms, but not in the opposite direction. Further, we infer that Soay and Sarda sheep carry introgressed mouflon alleles involved in bitter taste perception and/or innate immunity. Our results illustrate the potential for adaptive introgression even among recently diverged populations.

Expansion of alien carnivore and ungulate species in SW Poland

Changes in distribution of six mammal species, viz. Procyon lotor, Nyctereutes procyonoides, Neovison vison, Cervus nippon, Dama dama, Ovis aries musimon in SW Poland (42000 km2), since their introduction till 2008, have been analyzed. A rough estimate of their numbers is also provided for seven regions in SW Poland (Silesia). Detailed maps of distribution of the American mink and fallow deer in SW Poland during 2004–2008 are also presented. The alien ungulates, as long-established species in SW Poland, are not considered as particularly destructive to indigenous flora and fauna. They are not invasive, and have never been regarded as such. On the other hand, the alien carnivores are regarded in SW Poland as destructive to the indigenous fauna. Counter-measures for the rapidly increasing carnivores are proposed.

Tear production, intraocular pressure and conjunctival bacterial flora in selected captive wild ruminants.

Evaluation of tear production (Schirmer's tear test, STT) and measurement of intraocular pressure (IOP) were performed in a population of captive wild ungulates in a Slovenian ZOO during routine annual health check. In total, 10 fallow deer (Dama dama), 25 mouflons (Ovis aries musimon), 20 alpine ibexes (Capra ibex), and three alpine chamois (Rupicapra rupicapra) were included in the study. Tear production was performed by Schirmer's tear test, IOP was measured with an applanation tonometer, and ophthalmological examination was conducted with slit-lamp biomicroscopy and indirect ophthalmoscopy. Conjunctival swabs were taken and submitted for aerobic bacteriology and for detection of Chlamydia spp. and Mycoplasma spp. tested by PCR. Average tear production (in mm/min) was 17.8 ± 3.16 for fallow deer, 17.9 ± 3.87 for mouflons, and 11.7 ± 3.87 for ibexes. Mean intraocular pressure (IOP, in mm Hg) was 14.1 ± 2.48 for fallow deer, 14.9 ± 2.20 for mouflons, and 13.1 ± 2.43 for ibexes. For chamois, average tear production and IOP were 14.5 ± 3.0 and 10.2 ± 2.5, respectively; this is the first record of STT I and IOP in chamois. Bacteriological swabs were positive for bacteria in 100% of the fallow deer, 56% of mouflons, 35% of ibexes, and 100% of chamois. Gram-positive bacteria were predominant. Moraxella spp., Chlamydia spp., and Mycoplasma spp. were not detected. The reported values were obtained in animals under manual restraint only to be applicative in similar conditions.

Gastrointestinal nematodes and the deworming of mouflon (Ovis aries musimon) from Goleniowska Forest in West Pomerania province, Poland

Ruminants often live in environments where the natural balance has been disturbed by humans. As a result, there is a transfer of parasitosis to domestic animals and sometimes humans. The aim of the study was to determine the annual species composition and level of gastrointestinal nematode infection of mouflon (Ovis aries musimon) from the half-open breeding colonizing areas of the Goleniowska Forest, and to assess the effectiveness of deworming. The course of the parasitic infection of mouflon depends largely on geoclimatic and breeding factors. These diseases are caused by more than 17 species of nematodes with varying degrees of pathogenicity. Due to the high degree of parasitic infections in deer, both in Poland and abroad, it is necessary to develop specific preventive programs using preparations with a broad spectrum of action.

Spatio-temporal trends and risk factors affecting West Nile virus and related flavivirus exposure in Spanish wild ruminants.

BackgroundDuring the last decade, the spread of many flaviviruses (Genus Flavivirus) has been reported, representing an emerging threat for both animal and human health. To further study utility of wild ruminant samples in West Nile virus (WNV) surveillance, we assessed spatio–temporal trends and factors associated with WNV and cross-reacting flaviviruses exposure, particularly Usutu virus (USUV) and Meaban virus (MBV), in wild ruminants in Spain. Serum samples from 4693 wild ruminants, including 3073 free-living red deer (Cervus elaphus), 201 fallow deer (Dama dama), 125 mouflon (Ovis aries musimon), 32 roe deer (Capreolus capreolus) and 1262 farmed red deer collected in 2003–2014, were screened for WNV and antigenically-related flavivirus antibodies using a blocking ELISA (bELISA). Positive samples were tested for neutralizing antibodies against WNV, USUV and MBV by virus micro-neutralization tests.ResultsMean flavivirus seroprevalence according to bELISA was 3.4 ± 0.5 % in red deer, 1.0 ± 1.4 % in fallow deer, 2.4 ± 2.7 % in mouflon and 0 % in roe deer. A multivariate logistic regression model revealed as main risk factors for seropositivity in red deer; year (2011), the specific south-coastal bioregion (bioregion 5) and presence of wetlands. Red deer had neutralizing antibodies against WNV, USUV and MBV.ConclusionsThe results indicate endemic circulation of WNV, USUV and MBV in Spanish red deer, even in areas without known flavivirus outbreaks. WNV antibodies detected in a free-living red deer yearling sampled in 2010, confirmed circulation this year. Co-circulation of WNV and USUV was detected in bioregions 3 and 5, and of WNV and MBV in bioregion 3. Sampling of hunted and farmed wild ruminants, specifically of red deer yearlings, could be a complementary way to national surveillance programs to monitor the activity of emerging flaviviruses.

Wide exposure to Coxiella burnetii in ruminant and feline species living in a natural environment: zoonoses in a human-livestock-wildlife interface.

Assessment of the role of wild and domestic hosts as potential reservoirs of misdiagnosed zoonoses, such as Q fever by Coxiella burnetii, is an important public health issue today both for wildlife conservation and management of disease in human-livestock-wildlife interface. This study used ELISA, an indirect antibody, to research (2003-2013) C. burnetii infection in seven free-living wild and domestic ruminant species and in European wildcats (Felis silvestris). The animals studied were 0 European wildcats, 21 Spanish ibex (Capra pyrenaica), 314 red deer (Cervus elaphus), 556 fallow deer (Dama dama), 211 European mouflon (Ovis aries musimon), eight roe deer (Capreolus capreolus), 407 bovines (Bos taurus) and 3739 sheep (Ovis aries). All the animals shared the same habitat in the Serranía de Cuenca Natural Park (Castile-La Mancha, Spain). The study area is an example of human-domestic-wildlife interface where people and domestic animals live in close proximity to wildlife. Observed C. burnetii seropositive frequencies were: 33·3% European wildcats, 23·8% Spanish ibex, 22·5% domestic sheep 1·5% red deer, 1·4% European mouflon, 0·24% cattle, 0·18% fallow deer and 0% roe deer. The study found a wide C. burnetii prevalence of previous and present exposure in wild and domestic ruminant hosts in the Serranía de Cuenca Natural Park and reports the first evidence of C. burnetii exposure in free-living European wildcats.

Effect of winter conditions on wild ungulates mortality in the Owl Mountains (Poland)

Abstract Relations between climatic factors during the winter season (n days with frost &lt;0oC and −10oC; n days with snow cover: &gt;1 cm, &gt;10 cm and &gt;30 cm; maximum snow cover during season [cm]) and mortality in free-living ungulates (red deer Cervus elaphus Linnaeus, 1758, roe deer Capreolus capreolus Linnaeus, 1758 and mouflon Ovis aries musimon Linnaeus, 1758) in the Owl Mountains (Lower Silesia – Poland) in years 1998–2010 were investigated. Significant effects of all analysed climatic factors on ungulates mortality were documented. Correlations (Pearson) between such weather factors as the depth of snow cover and number of days with frost and recorded mortality in total animal populations analysed ranged from r = 0.33 to r = 0.77. The least adapted to local weather conditions was mouflon introduced to this area ca. 100 years ago from more southern parts of Europe. Roe deer species seems to be environmentally plastic, and are doing quite well in severe winters. Tolerance of red deer to the snow cover is much lower at low temperatures due to the fact that this species, during the period of snow cover, has limited access to the plants covered with snow and difficult access to food base.

Comparative histology of the femur between mouflon (Ovis aries musimon) and sheep (Ovis aries aries)

Mouflon (&lt;em&gt;Ovis aries musimon&lt;/em&gt;) and sheep (&lt;em&gt;Ovis aries aries&lt;/em&gt;) are considered as the wild and domestic subspecies of the same species. A comparative study on the microstructure of mouflon and sheep femoral bone diaphysis is here reported. Bone microstructure is described for the first time in the mouflon. More than 200 secondary osteons from both subspecies were analyzed and qualitative evaluation was followed by quantitative determination of perimeter, area, minimum and maximum diameters of secondary osteons and Haversian canals. The basic structural patterns observed in both subspecies can be classified as plexiform and irregular Haversian tissue, in accordance with what reported in the literature for most ruminants. The presence of many secondary osteons in the mouflon means that the bone also consists of dense Haversian bone tissue. Statistical analysis demonstrated that mouflon secondary osteons are larger than in the sheep and made of a greater number of lamellae. Since mouflon and sheep are taxonomically closely related and their body size is very similar, the qualitative and quantitative differences here reported could be primarily explained on account of their different lifestyle. Indeed, the habits of wildlife typical of mouflons may lead to the presence of wide areas of dense Haversian tissue in that subspecies, as mechanical stresses are known to be related to number and size of secondary osteons. Finally, this analysis could provide a useful tool to recognize bones from different species, in forensic exam and archaeozoological studies as well.

The role of wildlife in bluetongue virus maintenance in Europe: Lessons learned after the natural infection in Spain

Bluetongue (BT) is a re-emergent vector-borne viral disease of domestic and wild ruminants caused by bluetongue virus (BTV), a member of the genus Orbivirus. A complex multi-host, multi-vector and multi-pathogen (26 serotypes) transmission and maintenance network has recently emerged in Europe, and wild ruminants are regarded as an important node in this network. This review analyses the reservoir role of wild ruminants in Europe, identifying gaps in knowledge and proposing actions. Wild ruminant species are indicators of BTV circulation. Excepting the mouflon (Ovis aries musimon), European wild ungulates do not develop clinical disease. Diagnostic techniques used in wildlife do not differ from those used in domestic ruminants provided they are validated. Demographic, behavioural and physiological traits of wild hosts modulate their relationship with BTV vectors and with the virus itself. While BTV has been eradicated from central and northern Europe, it is still circulating in the Mediterranean Basin. We propose that currently two BTV cycles coexist in certain regions of the Mediterranean Basin, a wild one largely driven by deer of the subfamily Cervinae and a domestic one. These are probably linked through shared Culicoides vectors of several species. We suggest that wildlife might be contributing to this situation through vector maintenance and virus maintenance. Additionally, differences in temperature and other environmental factors add complexity to the Mediterranean habitats as compared to central and northern European ones. Intervention options in wildlife populations are limited. There is a need to know the role of wildlife in maintaining Culicoides populations, and to know which Culicoides species mediate the wildlife–livestock–BTV transmission events. There is also a clear need to study more in depth the links between Cervinae deer densities, environmental factors and BTV maintenance. Regarding disease control, we suggest that research efforts should be focused on wildlife population and wildlife disease monitoring.

Mycoplasma conjunctivae in domestic small ruminants from high mountain habitats in Northern Spain.

BackgroundInfectious keratoconjunctivitis (IKC) is a clinical condition affecting eyes of domestic and wild Caprinae worldwide, and Mycoplasma conjunctivae is considered the primary causative agent of IKC in sheep, goats and wild Caprinae. Domestic ruminants from high mountain habitats share grazing areas with wild mountain ungulates, such as chamois (Rupicapra spp.), Alpine ibex (Capra ibex) and European mouflon (Ovis aries musimon), and domestic sheep seem to act as M. conjunctivae reservoir. In this study, the presence of M. conjunctivae in domestic sheep and goats from the two main mountain ranges of Northern Spain, the Pyrenees and the Cantabrian Mountains, has been investigated.ResultsEye swabs were obtained from 439 domestic small ruminants selected from flocks that seasonally graze in alpine meadows during three consecutive years (2011-2012-2013). Seventy-nine out of the 378 domestic sheep (20.9%) tested positive to a M. conjunctivae specific real time-PCR (rt-PCR) in at least one eye, whereas all the 61 sampled domestic goats were negative. Statistically significant higher prevalence and higher proportion of infected flocks (P < 0.001) was observed in the Pyrenees (25.7%; 12 flocks out of 13), where M. conjunctivae is widespread and probably endemic in domestic sheep, than in the Cantabrian Mountains (7.8%; one flock out of six). Twenty-five sheep (three from the Pyrenees and 22 from the Cantabrian Mountains) which showed clinical signs consistent with infectious keratoconjunctivitis (IKC) were negative by rt-PCR. In contrast, 62 out of the 71 (87.3%) M. conjunctivae-positive sheep from the Pyrenees and the eight positive sheep from the Cantabrian Mountains were asymptomatic.ConclusionsThis study provides rt-PCR-based evidences of M. conjunctivae maintenance in domestic sheep, as well as a relationship between prevalence in domestic sheep and previously reported M. conjunctivae and IKC in wild ruminants. Domestic goats do not seem to play an important role in the epidemiology of M. conjunctivae in alpine habitats from Northern Spain.

Multilevel D-loop PCR identification of hunting game

The control region of mtDNA (D-loop) was used for hair samples of the five hunting game species identification: red deer (Cervus elaphus), roe deer (Capreolus capreolus), fallow deer (Dama dama), mouflon (Ovis aries musimon), and wild boar (Sus scrofa). For D-loop multilevel PCR detection scheme was applied in six primers (CE CVZV 1=5′-GATCACGAGCTTGATCACCA-3′; CE CVZV 2=5′-AGGAGTGGGCGATTTTAGGT-3′; DD CVZV 3=5′-CGCGTGAAACCAACAACCCGC-3′; DD CVZV 4=5′-CCGGGTCGGGGCCTTAGACG-3′; SSW CVZV 5=5′-ACACGTGCGTACACGCGCATA-3′; SSW CVZV 6=5′-GGTGCCTGCT T TCGTAGCACG-3′) designed to identify unknown biological samples of the hunting game animals. The PCR reaction volume was 25μl at conditions 95°C for 2min, 94°C for 30s, 60°C for 30s, 72°C for 30s, 35cycles, with last extension at 72°C for 10min. D-loop mtDNA amplicons of the game animals are characterized with specific PCR product sizes depending on species: red deer=163bp and 140bp, fallow deer=280bp and 138bp, roe deer=303bp, 280bp, 160bp and 138bp, mouflon=299bp and 178bp, wild boar=137bp and 229bp.

Detection of bluetongue serotype 4 in mouflons (Ovis aries musimon) from Spain

Bluetongue serotype 4 (BTV4) has been detected for the first time in tissue samples from 2 mouflons (Ovis aries musimon) from the South of Spain, in a retrospective study. The samples included in this study had been fixed and paraffin-embedded for over a year prior to their analysis using a BTV group-specific and a BTV4-specific RT-PCR test. Lung and lymphatic nodes were found positive in both specimens. The amplified DNA was confirmed to be BTV4 by sequencing the RT-PCR products and comparing them with other sequences from GenBank. The combination of RNA extraction from paraffin-embedded samples and serotype-specific real-time RT-PCR assays provides the tools for the detection of BTV from samples stored for a long time. The results shown in this study set out the basis for a greater survey with fixed samples from different species of wild ruminants that the veterinary services have been collecting for years.

The sequence and phylogenesis of the α-globin genes of Barbary sheep ( Ammotragus lervia), goat ( Capra hircus), European mouflon ( Ovis aries musimon) and Cyprus mouflon ( Ovis aries ophion)

In order to investigate the polymorphism of α-globin chain of hemoglobin amongst caprines, the linked Iα and IIα globin genes of Barbary sheep ( Ammotragus lervia), goat ( Capra hircus), European mouflon ( Ovis aries musimon), and Cyprus mouflon ( Ovis aries ophion) were completely sequenced, including the 5′ and 3′ untranslated regions. European and Cyprus mouflons, which do not show polymorphic α globin chains, had almost identical α globin genes, whereas Barbary sheep exhibit two different chains encoded by two nonallelic genes. Four different α genes were observed and sequenced in goat, validating previous observations of the existence of allelic and nonallelic polymorphism. As in other vertebrates, interchromosomal gene conversion appears to be responsible for such polymorphism. Evaluation of nucleotide sequences at the level of molecular evolution of the Iα-globin gene family in the caprine taxa suggests a closer relationship between the genus Ammotragus and Capra. Molecular clock estimates suggest sheep-mouflon, goat-aoudad, and ancestor-caprine divergences of 2.8, 5.7, and 7.1 MYBP, respectively.