Distal limb osteoarthritis in the horse

The aim of this thesis was to study two objective methods of osteoarthritis (OA) diagnosis in horses and use them on the assessment of new intra-articular treatments. The studied methods were a new inertial-sensor based system of lameness detection and cartilage biomarkers in serum. It was found that distal limb flexion is significantly correlated to the presence of metacarpo-phalangeal OA in hind limbs and that inertial-sensors are sensitive in detecting asymmetry in these cases. A positive and significant correlation was observed between Coll2-1 concentration in serum and the presence of joint disease in males and young horses. Fib3-2 measurement has good potential to be used since it is not influenced by sex or age. Using an experimental model of OA, adipose stem cells pre-activated with interferon-gamma decreased joint inflammation and radiographic lesions. In clinical cases, a single injection of high-concentrated and high-molecular weight hyaluronic-acid decreased joint inflammation and biomarkers’ concentration; OSTEOARTRITE DO MEMBRO DISTAL NO CAVALO Resumo: A finalidade desta tese foi estudar dois métodos de diagnóstico objetivo de osteoartrite (OA) em equinos e aplicá-los na avaliação de novas terapias intra-articulares. Utilizou-se um sistema de sensores de movimento e foi avaliada a concentração de biomarcadores de cartilagem no soro. Concluiu-se que a flexão distal positiva está correlacionada com OA na articulação metacarpofalângica nos membros posteriores e que os sensores são sensíveis na detecção de assimetria nestes casos. Existe uma correlação positiva e significativa entre as concentrações de Coll2-1 e a presença de doença articular, sobretudo em machos e jovens. A dosagem de Fib3-2 tem utilidade por não ser influenciada pelo sexo nem idade. Num modelo experimental da doença, a terapia à base de células estaminais reduziu a inflamação articular e as lesões radiográficas. Em casos clínicos, o tratamento com ácido-hialurónico de alta concentração e peso molecular provoca uma diminuição da inflamação articular e dos biomarcadores no soro.

Favourable areas for expansion and reintroduction of Iberian lynx accounting for distribution trends and genetic diversity of the European rabbit

Although on a local scale Iberian lynx distribution is determined by the availability of prey rabbits, recent modelling analyses have uncovered broad-scale disagreements between these two species’ distribution trends. These analyses showed also that the lynx had become restricted to only a fraction of the rabbit’s genetic diversity, and that this could be jeopardising its survival in the face of environmental hazards and uncertainty. In the present paper, a follow-up was carried out through the building of lynx and rabbit distribution models based on the most recent Spanish mammal atlas. Environmental favourability values for lynx and rabbit were positively correlated within the lynx’s current distribution area, but they were negatively correlated within the total Spanish area where lynx used to occur in the 1980’s. Environmental favourability for rabbits was significantly higher where lynx maintains reproductive populations than where it recently disappeared, indicating that rabbit favourability plays an important role and can be a good predictor of lynx persistence. The lynx and rabbit models were extrapolated to predict favourable areas for both species in Spain as well as in Portugal, on the original scale of the distribution data (10x10 km) and on a 100 times finer spatial resolution (1x1 km). The lynx and rabbit models were also combined through fuzzy logic to forecast the potential for lynx occurrence incorporating information on favourable areas for its main prey. Several areas are proposed as favourable for lynx expansion or re-introduction,

Conservation biogeography of ecologically interacting species: The case of the Iberian lynx and the European rabbit

Aim To relate the recent Iberian lynx decline to changes in the distribution of the European rabbit after the haemorrhagic disease outbreak of 1989. As Iberian rabbits evolved in two geographically separated lineages, being the recent lynx range practically restricted to the southwestern lineage, we also test if differential range dynamics exists for these lineages, with the consequent implications for lynx conservation and reintroduction planning.

Distribution modelling of wild rabbit hunting yields in its original area (S Iberian Peninsula)

In this work we used the information of the Annual Hunting Reports (AHRs) to obtain a high-resolution model of the potential favourableness for wild rabbit harvesting in Andalusia (southern Spain), using environmental and land-use variables as predictors. We analysed 32,134 AHRs from the period 1993/2001 reported by 6049 game estates to estimate the average hunting yields of wild rabbit in each Andalusian municipality (n5771). We modelled the favourableness for obtaining good hunting yields using stepwise logistic regression on a set of climatic, orographical, land use, and vegetation variables. The favourability equation was used to create a downscaled image representing the favourableness of obtaining good hunting yields for the wild rabbit in 161 km squares in Andalusia, using the Idrisi Image Calculator. The variables that affected hunting yields of wild rabbit were altitude, dry wood crops (mainly olive groves, almond groves, and vineyards), temperature, pasture, slope, and annual number of frost days. The 161 km squares with high favourableness values are scattered throughout the territory, which seems to be caused mainly by the effect of vegetation. Finally, we obtained quality categories for the territory by combining the probability values given by logistic regression with those of the environmental favourability function.

Relationship between biomarkers of cartilage in serum and degenerative joint disease in lusitanian horses

Cartilage degradation biomarkers are a potential tool for early diagnosis of degen- erative joint disease (DJD). In young horses, Coll2-1 and Coll2-1NO2 have been studied in serum and reported to be useful in the assessment of joint disease. Fib3-2 has been described to be higher in serum of humans with osteoarthritis but has not been assessed in horses. The aim of the current study was to evaluate biomarkers’ changes with age, sex, and exercise and correlate them with DJD. Blood collection and radiographic examination were performed in 51 Lusitanian horses. Moreover, inertial sensor-based detection of lameness was used to assess pain together with sub- jective examination. Females presented significantly higher concentrations of Coll2- 1 (P5.015) and Coll2-1NO2 (P5.014) compared to males. We found significant influence of high level of work in lower concentration of Coll2-1 (P5.001) and sig- nificant influence of sex in concentration of Coll2-1NO2 (P5.030). There was no influence of sex, age and work on Fib3-2. All biomarkers were increased in the DJD group (n535) compared to healthy controls (n516). This difference was significant for Coll2-1 (P5.015). When sorted by sex and age groups, significant difference in Coll2-1 between disease and healthy controls disappeared in old horses and females. Coll2-1 is a good marker of cartilage degradation in horses with DJD, being more specific in young horses and males. Fib3-2 may be further explored to help identify disease in particular cases.

A real time Taqman RT-PCR for the detection of rabbit hemorrhagic disease virus 2 (RHDV2)

As key prey, the wild rabbit downsize constitutes a major drawback on the endangered Iberian lynx (Lynx pardinus) re-introduction in the Iberia. Several captive breeding units mostly located in Alentejo, endeavour the wild rabbit repopulation of depleted areas assigned for the lynx re-introduction. Here we report an RHDV2 outbreak that occurred in early 2016 in a wild rabbit captive breeding unit located in Barrancos municipality. The estimated mortality rate between March and April 2016 was approximately 8.67%. Anatomopathologic examination was carried out for 13 victimized rabbits. Molecular characterization was based on the complete vp60 capsid gene. The 13 rabbit carcasses investigated showed typical macroscopic RHD lesions testing positive to RHDV2- RNA. Comparison of the vp60 nucleotide sequences obtained from two specimens with others publically available disclosed similarities below 98.22% with RHDV2 strains originated in the Iberia and Azores and revealed that the two identical strains from Barrancos-2016 contain six unique single synonymous nucleotide polymorphisms. In the phylogenetic analysis performed, the Barrancos-2016 strains clustered apart from other known strains, meaning they may represent new evolutionary RHDV2 lineages. No clear epidemiological link could be traced for this outbreak where the mortalities were lower compared with previous years. Yet, network analysis suggested a possible connection between the missing intermediates from which the strains from Barrancos 2013, 2014 and 2016 have derived. It is therefore possible that RHDV2 has circulated endemically in the region since 2012, with periodic epizootic occurrences. Still, six years after its emergence in wild rabbits, RHDV2 continues to pose difficulties to the establishment of natural wild rabbit populations that are crucial for the self-sustainability of the local ecosystems.

Relationship between biomarkers of cartilage in serum and degenerative joint disease in Lusitano horses

Introduction: Cartilage degradation biomarkers are a potential tool for early diagnosis of degenerative joint disease (DJD). In young horses, Coll2-1 and Coll2-1NO2 have been studied in serum and reported to be useful in the assessment of joint disease. Fib3-2 has been described to be higher in serum of humans with osteoarthritis but was never assessed in horses. The aim of the current study was to evaluate biomarkers’ changes with age, sex and exercise and correlate them with DJD. Material and Methods: Blood collection and radiographic examination were performed in 51 Lusitanian horses. Moreover, inertial sensor-based detection of lameness was used to assess pain together with subjective examination. Results: Females presented significantly higher concentrations of Coll2-1 (p = 0.015) and Coll2-1NO2 (p = 0.014) compared to males. We have found significant influence of high level of work in lower concentration of Coll2-1 (p = 0.001) and significant influence of sex in concentration of Coll2-1NO2 (p = 0.030). There was no influence of sex, age and work on Fib3-2. All biomarkers were increased in the DJD group (n= 35) compared to healthy controls (n = 16). This difference was significant for Coll2-1 (p = 0.015). When sorted by sex and age groups, significant difference in Coll2-1 between disease and healthy controls disappeared in old horses and females. Discussion/ Conclusion: Coll2-1 is a good marker of cartilage degradation in horses with DJD, being more specific in young horses and males. Fib3-2 may be further explored to help identify disease in particular cases.

Tracking the Origin of a Rabbit Haemorrhagic Virus 2 Outbreak in a Wild Rabbit Breeding Centre in Portugal; Epidemiological and Genetic Investigation

As key prey, the wild rabbit downsize constitutes a major drawback on the endangered Iberian lynx (Lynx pardinus) re-introduction in the Iberia. Several captive breeding units mostly located in Alentejo, endeavour the wild rabbit repopulation of depleted areas assigned for the lynx re-introduction. Here we report an RHDV2 outbreak that occurred in early 2016 in a wild rabbit captive breeding unit located in Barrancos municipality. The estimated mortality rate between March and April 2016 was approximately 8.67%. Anatomopathologic examination was carried out for 13 victimized rabbits. Molecular characterization was based on the complete vp60 capsid gene. The 13 rabbit carcasses investigated showed typical macroscopic RHD lesions testing positive to RHDV2-RNA. Comparison of the vp60 nucleotide sequences obtained from two specimens with others publically available disclosed similarities below 98.22% with RHDV2 strains originated in the Iberia and Azores and revealed that the two identical strains from Barrancos-2016 contain six unique single synonymous nucleotide polymorphisms. In the phylogenetic analysis performed, the Barrancos-2016 strains clustered apart from other known strains,meaning they may represent new evolutionary RHDV2 lineages. No clear epidemiological link could be traced for this outbreak where the mortalities were lower compared with previous years. Yet, network analysis suggested a possible connection between the missing intermediates from which the strains from Barrancos 2013, 2014 and 2016 have derived. It is therefore possible that RHDV2 has circulated endemically in the region since 2012, with periodic epizootic occurrences. Still, six years after its emergence in wild rabbits, RHDV2 continues to pose difficulties to the establishment of natural wild rabbit populations that are crucial for the self-sustainability of the local ecosystems.

Tracking the Origin of a Rabbit Haemorrhagic Virus 2 Outbreak in a Wild Rabbit Breeding Centre in Portugal; Epidemiological and Genetic Investigation

As key prey, the wild rabbit downsize constitutes a major drawback on the endangered Iberian lynx (Lynx pardinus) re-introduction in the Iberia. Several captive breeding units mostly located in Alentejo, endeavour the wild rabbit repopulation of depleted areas assigned for the lynx re-introduction. Here we report an RHDV2 outbreak that occurred in early 2016 in a wild rabbit captive breeding unit located in Barrancos municipality. The estimated mortality rate between March and April 2016 was approximately 8.67%. Anatomopathologic examination was carried out for 13 victimized rabbits. Molecular characterization was based on the complete vp60 capsid gene. The 13 rabbit carcasses investigated showed typical macroscopic RHD lesions testing positive to RHDV2- RNA. Comparison of the vp60 nucleotide sequences obtained from two specimens with others publically available disclosed similarities below 98.22% with RHDV2 strains originated in the Iberia and Azores and revealed that the two identical strains from Barrancos-2016 contain six unique single synonymous nucleotide polymorphisms. In the phylogenetic analysis performed, the Barrancos-2016 strains clustered apart from other known strains, meaning they may represent new evolutionary RHDV2 lineages. No clear epidemiological link could be traced for this outbreak where the mortalities were lower compared with previous years. Yet, network analysis suggested a possible connection between the missing intermediates from which the strains from Barrancos 2013, 2014 and 2016 have derived. It is therefore possible that RHDV2 has circulated endemically in the region since 2012, with periodic epizootic occurrences. Still, six years after its emergence in wild rabbits, RHDV2 continues to pose difficulties to the establishment of natural wild rabbit populations that are crucial for the self-sustainability of the local ecosystems.

Challenges in the rabbit haemorrhagic disease 2 (RHDV2) molecular diagnosis of vaccinated rabbits

Molecular methods are fundamental tools for the diagnosis of viral infections. While interpretation of results is straightforward for unvaccinated animals, where positivity represents ongoing or past infections, the presence of vaccine virus in the tissues of recently vaccinated animals may mislead diagnosis. In this study, we investigated the interference of RHDV2 vaccination in the results of a RT-qPCR for RHDV2 detection, and possible associations between mean Cq values of five animal groups differing in age, vaccination status and origin (domestic/wild). Viral sequences from vaccinated rabbits that died of RHDV2 infection (n = 14) were compared with the sequences from the commercial vaccines used in those animals. Group Cq means were compared through Independent t-test and One-way ANOVA. We proved that RHDV2 vaccine-RNA is not detected by the RT-qPCR as early as 15 days post- vaccination, an important fact in assisting results interpretation for diagnosis. Cq values of vaccinated and non-vaccinated infected domestic adults showed a statistically significant difference (p < 0.05), demonstrating that vaccination-induced immunity reduces viral loads and delays disease progression. Contrarily, in vaccinated young rabbits higher viral loads were registered compared to non-vaccinated kittens. No significant variation (p = 0.3824) was observed between viral loads of non- vaccinated domestic and wild RHDV2-victimised rabbits. Although the reduced number of vaccinated young animals analysed hampered a robust statistical analysis, this occurrence suggests that passively acquired maternal antibodies may inhibit the active immune response to vaccination, delaying protection and favouring disease progression. Our finding emphasises the importance of adapting kitten RHDV2 vaccination schedules to circumvent this interference phenomenon.