This page has only limited features, please log in for full access.
Commercial hare and rabbit immortalized cell lines are extremely limited regarding the many species within the lagomorpha order. To overcome this limitation, researchers and technicians must establish primary cell cultures derived from biopsies or embryos. Among all cell types, fibroblasts are plastic and resilient cells, highly convenient for clinical and fundamental research but also for diagnosis, particularly for viral isolation. Here, we describe a fast and cheap method to produce primary fibroblast cell cultures from leporid species, using dispase II, a protease that allows dermal–epidermal separation, followed by a simple enzymatic digestion with trypsin. This method allows for the establishment of an in vitro cell culture system with an excellent viability yield and purity level higher than 85% and enables the maintenance and even immortalization of leporid fibroblastic cells derived from tissues already differentiated.
Fábio A. Abade dos Santos; C. L. Carvalho; Isabel Almeida; Teresa Fagulha; Fernanda Rammos; Sílvia C. Barros; Margarida Henriques; Tiago Luís; Margarida D. Duarte. Simple Method for Establishing Primary Leporidae Skin Fibroblast Cultures. Cells 2021, 10, 2100 .
AMA StyleFábio A. Abade dos Santos, C. L. Carvalho, Isabel Almeida, Teresa Fagulha, Fernanda Rammos, Sílvia C. Barros, Margarida Henriques, Tiago Luís, Margarida D. Duarte. Simple Method for Establishing Primary Leporidae Skin Fibroblast Cultures. Cells. 2021; 10 (8):2100.
Chicago/Turabian StyleFábio A. Abade dos Santos; C. L. Carvalho; Isabel Almeida; Teresa Fagulha; Fernanda Rammos; Sílvia C. Barros; Margarida Henriques; Tiago Luís; Margarida D. Duarte. 2021. "Simple Method for Establishing Primary Leporidae Skin Fibroblast Cultures." Cells 10, no. 8: 2100.
In late 2019, the first herpesvirus in the genus Lepus, named leporid gammaherpesvirus 5 (LeHV-5) was described. At the time, herpetic typical lesions were observed in hares infected by the myxoma virus, which is known to induce immunosuppression. Though the real impact of LeHV-5 is still poorly understood, since it affects reproduction, it poses an additional threat to the already fragile populations of Iberian hare, demanding prevalence investigations. In this article, we describe the first quantitative molecular method for LeHV-5 detection, using either Taqman or the EvaGreen systems. This method has excellent sensitivity and specificity, it is able to detect 2.1 copies of LeHV-5 DNA and was validated with an internal control targeting the 18S rRNA gene, allowing monitoring extraction and PCR amplification efficiencies.
Fábio Abade dos Santos; Carina Carvalho; Maria Peleteiro; Francisco Parra; Margarida Duarte. A Versatile qPCR for Diagnosis of Leporid Gammaherpesvirus 5 Using Evagreen® or Taqman® Technologies. Viruses 2021, 13, 715 .
AMA StyleFábio Abade dos Santos, Carina Carvalho, Maria Peleteiro, Francisco Parra, Margarida Duarte. A Versatile qPCR for Diagnosis of Leporid Gammaherpesvirus 5 Using Evagreen® or Taqman® Technologies. Viruses. 2021; 13 (4):715.
Chicago/Turabian StyleFábio Abade dos Santos; Carina Carvalho; Maria Peleteiro; Francisco Parra; Margarida Duarte. 2021. "A Versatile qPCR for Diagnosis of Leporid Gammaherpesvirus 5 Using Evagreen® or Taqman® Technologies." Viruses 13, no. 4: 715.
Rabbit haemorrhagic disease (RHD) is a highly contagious infectious disease of European wild and domestic rabbits. Rabbit haemorrhagic disease virus (RHDV, GI.1) emerged in 1986 in Europe, rapidly spreading all over the world. Several genotypes of RHDV have been recognised over time, but in 2010, a new virus (RHDV2/RHDVb, GI.2) emerged and progressively replaced the previous RHDV strains, due to the lack of cross-immunity conferred between RHDV and RHDV2. RHDV2 has a high mutation rate, similarly to the other calivirus and recombines with strains of RHDV and non-pathogenic calicivirus (GI.4), ensuring the continuous emergence of new field strains. Although this poses a threat to the already endangered European rabbit species, the available vaccines against RHDV2 and the compliance of biosafety measures seem to be controlling the infection in the rabbit industry Pet rabbits, especially when kept indoor, are considered at lower risk of infections, although RHDV2 and myxoma virus (MYXV) constitute a permanent threat due to transmission via insects. Vaccination against these viruses is therefore recommended every 6 months (myxomatosis) or annually (rabbit haemorrhagic disease). The combined immunization for myxomatosis and RHDV through a commercially available bivalent vaccine with RHDV antigen has been extensively used (Nobivac® Myxo-RHD, MSD, Kenilworth, NJ, USA). This vaccine however does not confer proper protection against the RHDV2, thus the need for a rabbit clinical vaccination protocol update. Here we report a clinical case of hepatitis and alteration of coagulation in a pet rabbit that had been vaccinated with the commercially available bivalent vaccine against RHDV and tested positive to RHDV2 after death. The animal developed a prolonged and atypical disease, compatible with RHD. The virus was identified to be an RHDV2 recombinant strain, with the structural backbone of RHDV2 (GI.2) and the non-structural genes of non-pathogenic-A1 strains (RCV-A1, GI.4). Although confirmation of the etiological agent was only made after death, the clinical signs and analytic data were very suggestive of RHD.
Fábio A. Abade Dos Santos; Carolina Magro; Carina L. Carvalho; Pedro Ruivo; Margarida D. Duarte; Maria C. Peleteiro. A Potential Atypical Case of Rabbit Haemorrhagic Disease in a Dwarf Rabbit. Animals 2020, 11, 40 .
AMA StyleFábio A. Abade Dos Santos, Carolina Magro, Carina L. Carvalho, Pedro Ruivo, Margarida D. Duarte, Maria C. Peleteiro. A Potential Atypical Case of Rabbit Haemorrhagic Disease in a Dwarf Rabbit. Animals. 2020; 11 (1):40.
Chicago/Turabian StyleFábio A. Abade Dos Santos; Carolina Magro; Carina L. Carvalho; Pedro Ruivo; Margarida D. Duarte; Maria C. Peleteiro. 2020. "A Potential Atypical Case of Rabbit Haemorrhagic Disease in a Dwarf Rabbit." Animals 11, no. 1: 40.
In late 2018, an epidemic myxomatosis outbreak emerged on the Iberian Peninsula leading to high mortality in Iberian hare populations. A recombinant Myxoma virus (strains MYXV-Tol and ha-MYXV) was rapidly identified, harbouring a 2.8 kbp insertion containing evolved duplicates of M060L, M061L, M064L, and M065L genes from myxoma virus (MYXV) or other Poxviruses. Since 2017, 1616 rabbits and 125 hares were tested by a qPCR directed to M000.5L/R gene, conserved in MYXV and MYXV-Tol/ha-MYXV strains. A subset of the positive samples (20%) from both species was tested for the insert with MYXV being detected in rabbits and the recombinant MYXV in hares. Recently, three wild rabbits were found dead South of mainland Portugal, showing skin oedema and pulmonary lesions that tested positive for the 2.8 kbp insert. Sequencing analysis showed 100% similarity with the insert sequences described in Iberian hares from Spain. Viral particles were observed in the lungs and eyelids of rabbits by electron microscopy, and isolation in RK13 cells attested virus infectivity. Despite that the analysis of complete genomes may predict the recombinant MYXV strains’ ability to infect rabbit, routine analyses showed species segregation for the circulation of MYXV and recombinant MYXV in wild rabbit and in Iberian hares, respectively. This study demonstrates, however, that recombinant MYXV can effectively infect and cause myxomatosis in wild rabbits and domestic rabbits, raising serious concerns for the future of the Iberian wild leporids while emphasises the need for the continuous monitoring of MYXV and recombinant MYXV in both species.
Fábio A. Abade Dos Santos; Carina L. Carvalho; Andreia Pinto; Ranjit Rai; Madalena Monteiro; Paulo Carvalho; Paula Mendonça; Maria C. Peleteiro; Francisco Parra; Margarida D. Duarte. Detection of recombinant Hare Myxoma Virus in wild rabbits (Oryctolagus cuniculus algirus). Viruses 2020, 12, 1127 .
AMA StyleFábio A. Abade Dos Santos, Carina L. Carvalho, Andreia Pinto, Ranjit Rai, Madalena Monteiro, Paulo Carvalho, Paula Mendonça, Maria C. Peleteiro, Francisco Parra, Margarida D. Duarte. Detection of recombinant Hare Myxoma Virus in wild rabbits (Oryctolagus cuniculus algirus). Viruses. 2020; 12 (10):1127.
Chicago/Turabian StyleFábio A. Abade Dos Santos; Carina L. Carvalho; Andreia Pinto; Ranjit Rai; Madalena Monteiro; Paulo Carvalho; Paula Mendonça; Maria C. Peleteiro; Francisco Parra; Margarida D. Duarte. 2020. "Detection of recombinant Hare Myxoma Virus in wild rabbits (Oryctolagus cuniculus algirus)." Viruses 12, no. 10: 1127.
In late 2018, an epidemic myxomatosis outbreak emerged on the Iberian Peninsula leading to high mortality in Iberian hare populations. Soon, a recombinant virus (MYXV-Tol or ha-MYXV) was identified, harboring a 2.8 kb insertion containing evolved duplicates of M060L, M061L, M064L, and M065L from MYXV. Since 2017, 1616 rabbits and 82 hares were tested by a qPCR directed to M000.5L/R gene, conserved in MYXV and MYXV-Tol/ ha-MYXV strains. A subset (20%) of the positive samples was tested for the insert with MYXV being detected in rabbits and recombinant MYXV in hares. Recently, two wild rabbits found dead in South Portugal, showing skin oedema and pulmonary lesions tested positive for the 2.8 Kb insert. Sequencing showed 100% similarity with the insert sequences described in Iberian hares from Spain. Viral particles were observed in the lungs of both rabbits by electron microscopy, and isolation in RK13 cells showed virus infectivity. Despite the analysis of recombinant MYXV genomes may predict its ability to infect rabbit, routine analyses showed species segregation for the circulation of MYXV and recombinant MYXV in wild rabbit and in Iberian hares, respectively. This study demonstrates, however, that recombinant MYXV can effectively infect and cause myxomatosis in wild rabbits and domestic rabbits, which raises serious concerns for the future of the Iberian wild leporids and emphasizes the need to continue monitoring MYXV and recombinant MYXV in both species.
Fábio A. Abade Dos Santos; Carina L. Carvalho; Andreia Pinto; Ranjit Rai; Madalena Monteiro; Paulo Carvalho; Paula Mendonça; Maria C. Peleteiro; Francisco Parra; Margarida D. Duarte. Detection of Recombinant Hare Myxoma Virus in Wild Rabbits (Oryctolagus cuniculus algirus). 2020, 1 .
AMA StyleFábio A. Abade Dos Santos, Carina L. Carvalho, Andreia Pinto, Ranjit Rai, Madalena Monteiro, Paulo Carvalho, Paula Mendonça, Maria C. Peleteiro, Francisco Parra, Margarida D. Duarte. Detection of Recombinant Hare Myxoma Virus in Wild Rabbits (Oryctolagus cuniculus algirus). . 2020; ():1.
Chicago/Turabian StyleFábio A. Abade Dos Santos; Carina L. Carvalho; Andreia Pinto; Ranjit Rai; Madalena Monteiro; Paulo Carvalho; Paula Mendonça; Maria C. Peleteiro; Francisco Parra; Margarida D. Duarte. 2020. "Detection of Recombinant Hare Myxoma Virus in Wild Rabbits (Oryctolagus cuniculus algirus)." , no. : 1.
Myxomatosis was detected in Iberian hares (Lepus granatensis) in Portugal, October 2018, following its emergence in Spain 3 months earlier. Here, we describe the epidemiological, molecular and anatomo-histopathological data of the first two cases. Myxoma virus DNA was detected in the eyelids, nose and perineal region in both hares. It was also detected in the lungs of hare 1 and in the spleen and liver of hare 2. The genomic insertion identified in strains from Spain was confirmed in both strains suggesting a common origin for the Iberian viruses. Gross lesions in hare 1 included palpebral oedema and conjunctival mucopurulent discharge, common in both forms of the disease in rabbits. Hare 2 presented eyelid thickening with small diffuse nodules. Histopathology of the eyelids showed extracellular myxoid matrix in hare 1 and purulent dermatitis in hare 2. Both animals exhibited good body condition, suggesting a short course of the disease and higher virulence of the virus towards the Iberian hare.
Carina Luísa Carvalho; Fábio Alexandre Abade Dos Santos; Madelena Monteiro; Paulo Carvalho; Paula Mendonça; Margarida Dias Duarte. First cases of myxomatosis in Iberian hares ( Lepus granatensis ) in Portugal. Veterinary Record Case Reports 2020, 8, e001044 .
AMA StyleCarina Luísa Carvalho, Fábio Alexandre Abade Dos Santos, Madelena Monteiro, Paulo Carvalho, Paula Mendonça, Margarida Dias Duarte. First cases of myxomatosis in Iberian hares ( Lepus granatensis ) in Portugal. Veterinary Record Case Reports. 2020; 8 (2):e001044.
Chicago/Turabian StyleCarina Luísa Carvalho; Fábio Alexandre Abade Dos Santos; Madelena Monteiro; Paulo Carvalho; Paula Mendonça; Margarida Dias Duarte. 2020. "First cases of myxomatosis in Iberian hares ( Lepus granatensis ) in Portugal." Veterinary Record Case Reports 8, no. 2: e001044.
Myxoma virus (MYXV) and rabbit haemorrhagic disease virus 2 (RHDV2) are two major pathogens that affect the European rabbit (Oryctolagus cuniculus). Between August 2017 and August 2019, 1166 wild rabbits (971 legally hunted and 195 found dead) were tested by PCR-based methods for MYXV and RHDV2 within the scope of an ongoing surveillance programme on wild leporids in Portugal. Despite never having been reported before and being considered a rare event, coinfection by RHDV2 and MYXV was detected in one juvenile wild rabbit found dead in the Évora district located in Alentejo. The relative frequency of coinfection in the group of diseased rabbits (found dead in the field) was 0.52 per cent (1/195). The positivity percentage of each single virus was much higher, namely, 14.36 per cent (28/195) for MYXV and 55.38 per cent (108/195) for RHDV2, within the 2 years of sample collection considered.
Carina Luisa Carvalho; Fábio Alexandre Abade Dos Santos; Teresa Fagulha; Paulo Carvalho; Paula Mendonça; Madalena Monteiro; Margarida Dias Duarte. Myxoma virus and rabbit haemorrhagic disease virus 2 coinfection in a European wild rabbit ( Oryctolagus cuniculus algirus ), Portugal. Veterinary Record Case Reports 2020, 8, e001002 .
AMA StyleCarina Luisa Carvalho, Fábio Alexandre Abade Dos Santos, Teresa Fagulha, Paulo Carvalho, Paula Mendonça, Madalena Monteiro, Margarida Dias Duarte. Myxoma virus and rabbit haemorrhagic disease virus 2 coinfection in a European wild rabbit ( Oryctolagus cuniculus algirus ), Portugal. Veterinary Record Case Reports. 2020; 8 (1):e001002.
Chicago/Turabian StyleCarina Luisa Carvalho; Fábio Alexandre Abade Dos Santos; Teresa Fagulha; Paulo Carvalho; Paula Mendonça; Madalena Monteiro; Margarida Dias Duarte. 2020. "Myxoma virus and rabbit haemorrhagic disease virus 2 coinfection in a European wild rabbit ( Oryctolagus cuniculus algirus ), Portugal." Veterinary Record Case Reports 8, no. 1: e001002.
During the necropsies of Iberian hares obtained in 2018/2019, along with signs of the nodular form of myxomatosis, other unexpected external lesions were also observed. Histopathology revealed nuclear inclusion bodies in stromal cells suggesting the additional presence of a nuclear replicating virus.Transmission electron microscopy further demonstrated the presence of herpesvirus particles in the tissues of affected hares.We confirmed the presence of herpesvirus in 13 MYXV-positive hares by PCR and sequencing analysis. Herpesvirus-DNA was also detected in seven healthy hares, suggesting its asymptomatic circulation.Phylogenetic analysis based on DNA polymerase gene, grouped this virus within the gammaherpesviruses, close to herpesviruses from bats and rodents.We propose to name this new virus Leporid gammaherpesvirus 5 (LeHV-5), according to the International Committee on Taxonomy of Viruses standards.The impact of herpesvirus infection on the reproduction and mortality of the Iberian hare is yet unknown but may aggravate the decline of wild populations caused by the recently emerged natural recombinant myxoma virus.
F.A. Abade Dos Santos; Madalena Monteiro; Andreia Pinto; Carina L. Carvalho; M. C. Peleteiro; Paulo Carvalho; Paula Mendonça; Tânia Carvalho; Margarida D. Duarte. First description of a herpesvirus infection in genus Lepus. 2020, 1 .
AMA StyleF.A. Abade Dos Santos, Madalena Monteiro, Andreia Pinto, Carina L. Carvalho, M. C. Peleteiro, Paulo Carvalho, Paula Mendonça, Tânia Carvalho, Margarida D. Duarte. First description of a herpesvirus infection in genus Lepus. . 2020; ():1.
Chicago/Turabian StyleF.A. Abade Dos Santos; Madalena Monteiro; Andreia Pinto; Carina L. Carvalho; M. C. Peleteiro; Paulo Carvalho; Paula Mendonça; Tânia Carvalho; Margarida D. Duarte. 2020. "First description of a herpesvirus infection in genus Lepus." , no. : 1.
In the regular wildlife monitoring action carried out in the summer of the past few years at the Berlenga Island, wild rabbits (Oryctolagus cuniculus) have been repeatedly found dead. However, the origin of those deaths was never investigated. Our aim was to investigate the cause of death of 11 rabbits collected between April and May 2016. While screening samples from rabbit carcasses for the major viral rabbit pathogens, five tested positive to RHDV2 but all were negative for RHDV and myxoma virus (MYXV). For six RHDV2-negative specimens, emaciation and parasitism were considered the most probable cause of death. Lesions identified in the RHDV2-positive rabbits included non-suppurative diffuse hepatic necrosis and pulmonary lesions varying from congestion and oedema of the lungs to interstitial pneumonia. Sequencing analysis of the vp60 gene obtained from two specimens showed identical vp60 sequences. Comparison with other known RHDV2 strains from public databases through BLAST analysis revealed a closer similarity with strains from Alentejo collected during 2013. Maximum Likelihood and Bayesian phylogenetic analysis showed that the 2016 strains from the archipelago have a higher resemblance with a group of strains mostly collected in the South of Portugal between 2013 and 2014. The results suggest that RHDV2 may have been introduced on the Berlenga Island a few years ago, having evolved separately from mainland strains due to insularity.
F.A. Abade Dos Santos; C. Carvalho; Oliveira Nuno; J. J. Correia; M. Henriques; M. C. Peleteiro; M. Fevereiro; M. D. Duarte. Detection of rabbit Haemorrhagic disease virus 2 during the wild rabbit (Oryctolagus cuniculus) eradication from the Berlengas archipelago, Portugal. BMC Veterinary Research 2017, 13, 336 .
AMA StyleF.A. Abade Dos Santos, C. Carvalho, Oliveira Nuno, J. J. Correia, M. Henriques, M. C. Peleteiro, M. Fevereiro, M. D. Duarte. Detection of rabbit Haemorrhagic disease virus 2 during the wild rabbit (Oryctolagus cuniculus) eradication from the Berlengas archipelago, Portugal. BMC Veterinary Research. 2017; 13 (1):336.
Chicago/Turabian StyleF.A. Abade Dos Santos; C. Carvalho; Oliveira Nuno; J. J. Correia; M. Henriques; M. C. Peleteiro; M. Fevereiro; M. D. Duarte. 2017. "Detection of rabbit Haemorrhagic disease virus 2 during the wild rabbit (Oryctolagus cuniculus) eradication from the Berlengas archipelago, Portugal." BMC Veterinary Research 13, no. 1: 336.
We report the detection of rabbit haemorrhagic disease virus 2 (RHDV2) in the Madeira archipelago, Portugal. Viral circulation was confirmed by RT-qPCR and vp60 sequencing. Epidemiological data revealed the outbreak initiated in October 2016 in Porto Santo affecting wild and domestic rabbits. It was then detected three months later on the island of Madeira. Five haplotypes were identified and a genetic overall similarity of 99.54 to 99.89% was observed between the two viral populations. Unique single nucleotide polymorphisms were recognised in the Madeira archipelago strains, two of which resulting in amino acid substitutions at positions 480 and 570 in the VP60 protein. Phylogenetic investigation by Maximum Likelihood showed all the vp60 sequences from the Madeira archipelago group together with high bootstraps. The analysis also showed that the Madeira archipelago strains are closely related to the strains detected in the south of mainland Portugal in 2016, suggesting a possible introduction from the mainland. The epidemiological data and high genetic similarity indicate a common source for the Porto Santo and Madeira RHDV2 outbreaks. Human activity related to hunting was most probably at the origin of the Madeira outbreak.
Carina Luísa Carvalho; Sara Silva; Paz Gouveia; Margarida Costa; Elsa Leclerc Duarte; Ana Margarida Henriques; Sílvia Santos Barros; Tiago Luís; Fernanda Ramos; Teresa Fagulha; Miguel Fevereiro; Margarida Dias Duarte. Emergence of rabbit haemorrhagic disease virus 2 in the archipelago of Madeira, Portugal (2016–2017). Virus Genes 2017, 53, 922 -926.
AMA StyleCarina Luísa Carvalho, Sara Silva, Paz Gouveia, Margarida Costa, Elsa Leclerc Duarte, Ana Margarida Henriques, Sílvia Santos Barros, Tiago Luís, Fernanda Ramos, Teresa Fagulha, Miguel Fevereiro, Margarida Dias Duarte. Emergence of rabbit haemorrhagic disease virus 2 in the archipelago of Madeira, Portugal (2016–2017). Virus Genes. 2017; 53 (6):922-926.
Chicago/Turabian StyleCarina Luísa Carvalho; Sara Silva; Paz Gouveia; Margarida Costa; Elsa Leclerc Duarte; Ana Margarida Henriques; Sílvia Santos Barros; Tiago Luís; Fernanda Ramos; Teresa Fagulha; Miguel Fevereiro; Margarida Dias Duarte. 2017. "Emergence of rabbit haemorrhagic disease virus 2 in the archipelago of Madeira, Portugal (2016–2017)." Virus Genes 53, no. 6: 922-926.
Rabbit haemorrhagic disease virus 2 (RHDV2) emerged recently in several European countries, leading to extensive economic losses in the industry. In response to this new infection, specific inactivated vaccines were developed in Europe and full and rapid setup of protective immunity induced by vaccination was reported. However, data on the efficacy of these vaccines in an ongoing-infection scenario is unavailable. In this study we investigated an infected RHDV2 indoor industrial meat rabbitry, where fatalities continued to occur after the implementation of the RHDV2 vaccination, introduced to control the disease. The aim of this study was to understand if these mortalities were RHDV2-related, to discover if the dead animals showed any common features such as age or time distance from vaccination, and to identify the source of the outbreak. Anatomo-pathological analysis of vaccinated animals with the virus showed lesions compatible with systemic haemorrhagic disease and RHDV2-RNA was detected in 85.7% of the animals tested. Sequencing of the vp60 gene amplified from liver samples led to the recognition of RHDV2 field strains demonstrating that after the implementation of vaccination, RHDV2 continued to circulate in the premises and to cause sporadic deaths. A nearby, semi-intensive, RHDV2 infected farm belonging to the same owner was identified as the most probable source of the virus. The main risk factors for virus introduction in these two industries were identified. Despite the virus being able to infect a few of the vaccinated rabbits, the significant decrease in mortality rate observed in vaccinated adult rabbits clearly reflects the efficacy of the vaccination. Nonetheless, the time taken to control the infection also highlights the importance of RHDV2 vaccination prior to the first contact with the virus, highly recommendable in endemic areas, to mitigate the infection’s impact on the industry.
Carina Carvalho; Elsa Leclerc Duarte; J.M. Monteiro; C. Afonso; J. Pacheco; P. Carvalho; P. Mendonça; A. Botelho; T. Albuquerque; P. Themudo; M. Fevereiro; A.M. Henriques; Silvia Barros; M. Dias Duarte. Progression of rabbit haemorrhagic disease virus 2 upon vaccination in an industrial rabbitry: a laboratorial approach. World Rabbit Science 2017, 25, 73 -85.
AMA StyleCarina Carvalho, Elsa Leclerc Duarte, J.M. Monteiro, C. Afonso, J. Pacheco, P. Carvalho, P. Mendonça, A. Botelho, T. Albuquerque, P. Themudo, M. Fevereiro, A.M. Henriques, Silvia Barros, M. Dias Duarte. Progression of rabbit haemorrhagic disease virus 2 upon vaccination in an industrial rabbitry: a laboratorial approach. World Rabbit Science. 2017; 25 (1):73-85.
Chicago/Turabian StyleCarina Carvalho; Elsa Leclerc Duarte; J.M. Monteiro; C. Afonso; J. Pacheco; P. Carvalho; P. Mendonça; A. Botelho; T. Albuquerque; P. Themudo; M. Fevereiro; A.M. Henriques; Silvia Barros; M. Dias Duarte. 2017. "Progression of rabbit haemorrhagic disease virus 2 upon vaccination in an industrial rabbitry: a laboratorial approach." World Rabbit Science 25, no. 1: 73-85.
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.
Carina Carvalho; Elsa Leclerc Duarte; M. Monteiro; Ana Botelho; T. Albuquerque; Miguel Fevereiro; A.M. Henriques; Silvia Barros; Margarida Duarte. Challenges in the rabbit haemorrhagic disease 2 (RHDV2) molecular diagnosis of vaccinated rabbits. Veterinary Microbiology 2016, 198, 43 -50.
AMA StyleCarina Carvalho, Elsa Leclerc Duarte, M. Monteiro, Ana Botelho, T. Albuquerque, Miguel Fevereiro, A.M. Henriques, Silvia Barros, Margarida Duarte. Challenges in the rabbit haemorrhagic disease 2 (RHDV2) molecular diagnosis of vaccinated rabbits. Veterinary Microbiology. 2016; 198 ():43-50.
Chicago/Turabian StyleCarina Carvalho; Elsa Leclerc Duarte; M. Monteiro; Ana Botelho; T. Albuquerque; Miguel Fevereiro; A.M. Henriques; Silvia Barros; Margarida Duarte. 2016. "Challenges in the rabbit haemorrhagic disease 2 (RHDV2) molecular diagnosis of vaccinated rabbits." Veterinary Microbiology 198, no. : 43-50.
The presence of Francisella species in 2134 ticks, 93 lagomorphs and 280 small mammals from the Iberian Peninsula was studied. Overall, 19 ticks and 6 lagomorphs were positive for Francisella tularensis subsp. holarctica, suggesting, as described for other regions, that lagomorphs may have an important role in the maintenance of F. tularensis in nature. Of the 6 positive lagomorphs, 4 were identified as the European rabbit, Oryctogalus cuniculus. Additionally, 353 ticks and 3 small mammals were PCR positive for Francisella-like endosymbionts (FLEs) and one small mammal was also positive for Francisella hispaniensis-like DNA sequences. Among FLE positive specimens, a variety of sequence types were detected: ticks were associated with 5 lpnA sequence types, with only one type identified per tick, in contrast to 2 lpnA sequence types detected in a single wood mouse (Apodemus sylvaticus). To our knowledge, this is the first report of FLEs in free-living small mammals as well as the first detection of F. hispaniensis-like sequences in a natural setting.This work was supported by Fundação para a Ciência e Tecnologia (FCT) project: PTDC/SAU-ESA/104947/2008 and a scholarship (SFRH/BD/79225/2011); and also by the Fondo deInvestigation Sanitaria (PI10/00165) and Instituto de Salud CarlosIII (grant EM03/06).info:eu-repo/semantics/publishedVersio
Isabel Lopes de Carvalho; A. Toledo; Carina Carvalho; J.F. Barandika; L.B. Respicio-Kingry; C. Garcia-Amil; Ana L. Garcia-Perez; A. Sonia Olmeda; Líbia Zé-Zé; J.M. Petersen; Pedro Anda; Maria Sofia Núncio; Raquel Escudero. Francisella species in ticks and animals, Iberian Peninsula. Ticks and Tick-borne Diseases 2016, 7, 159 -165.
AMA StyleIsabel Lopes de Carvalho, A. Toledo, Carina Carvalho, J.F. Barandika, L.B. Respicio-Kingry, C. Garcia-Amil, Ana L. Garcia-Perez, A. Sonia Olmeda, Líbia Zé-Zé, J.M. Petersen, Pedro Anda, Maria Sofia Núncio, Raquel Escudero. Francisella species in ticks and animals, Iberian Peninsula. Ticks and Tick-borne Diseases. 2016; 7 (1):159-165.
Chicago/Turabian StyleIsabel Lopes de Carvalho; A. Toledo; Carina Carvalho; J.F. Barandika; L.B. Respicio-Kingry; C. Garcia-Amil; Ana L. Garcia-Perez; A. Sonia Olmeda; Líbia Zé-Zé; J.M. Petersen; Pedro Anda; Maria Sofia Núncio; Raquel Escudero. 2016. "Francisella species in ticks and animals, Iberian Peninsula." Ticks and Tick-borne Diseases 7, no. 1: 159-165.
Carina Luisa Carvalho; Joana Rodeia; Sandra Branco; Ra Branco; Madalena Monteiro; Elsa Leclerc Duarte; Pedro Melo Patrícia Tavares Santos; Ana Margarida Henriques Mourão; Sílvia Santos Barros; A Ramos; Teresa Fagulha; Miguel Fevereiro; Margarida Dias Duarte. Tracking the Origin of a Rabbit Haemorrhagic Virus 2 Outbreak in a Wild Rabbit Breeding Centre in Portugal; Epidemiological and Genetic Investigation. Journal of Emerging Infectious Diseases 2016, 1, 1 .
AMA StyleCarina Luisa Carvalho, Joana Rodeia, Sandra Branco, Ra Branco, Madalena Monteiro, Elsa Leclerc Duarte, Pedro Melo Patrícia Tavares Santos, Ana Margarida Henriques Mourão, Sílvia Santos Barros, A Ramos, Teresa Fagulha, Miguel Fevereiro, Margarida Dias Duarte. Tracking the Origin of a Rabbit Haemorrhagic Virus 2 Outbreak in a Wild Rabbit Breeding Centre in Portugal; Epidemiological and Genetic Investigation. Journal of Emerging Infectious Diseases. 2016; 1 (4):1.
Chicago/Turabian StyleCarina Luisa Carvalho; Joana Rodeia; Sandra Branco; Ra Branco; Madalena Monteiro; Elsa Leclerc Duarte; Pedro Melo Patrícia Tavares Santos; Ana Margarida Henriques Mourão; Sílvia Santos Barros; A Ramos; Teresa Fagulha; Miguel Fevereiro; Margarida Dias Duarte. 2016. "Tracking the Origin of a Rabbit Haemorrhagic Virus 2 Outbreak in a Wild Rabbit Breeding Centre in Portugal; Epidemiological and Genetic Investigation." Journal of Emerging Infectious Diseases 1, no. 4: 1.
Rabbit haemorrhagic disease virus 2 (RHDV2) is widespread in several countries of Western Europe, but it has not been introduced to other continents. However, between late 2014 and early 2015, the presence of RHDV2 was confirmed outside of the European continent, in the Azores, initially in the islands of Graciosa, Flores, S. Jorge and Terceira. In this study we report the subsequent detection of RHDV2 in wild rabbits from the islands of Faial, St. Maria and S. Miguel, and display the necropsy and microscopic examination data obtained, which showed lesions similar to those induced by classical strains of RHDV, with severe affection of lungs and liver. We also disclose the result of a genetic investigation carried out with RHDV2 positive samples from wild rabbits found dead in the seven islands. Partial vp60 sequences were amplified from 27 tissue samples. Nucleotide analysis showed that the Azorean strains are closely related to each other, sharing a high genetic identity (>99.15%). None of the obtained sequences were identical to any RHDV2 sequence publically known, hampering a clue for the source of the outbreaks. However, Bayesian and maximum likelihood phylogenetic analyses disclosed that Azorean strains are more closely related to a few strains from Southern Portugal than with any others presently known. In the analysed region comprising the terminal 942 nucleotides of the vp60 gene, four new single nucleotide polymorphisms (SNP) were identified. Based on the present data, these four SNPs, which are unique in the strains from Azores, may constitute putative molecular geographic markers for Azorean RHDV2 strains, if they persist in the future. One of these variations is a non-synonymous substitution that involves the replacement of one amino acid in a hypervariable region of the capsid protein.
Margarida Duarte; Carina Carvalho; Susana Bernardo; Silvia Barros; Sandra Benevides; Lídia Flor; Madalena Monteiro; Maria Isabel Marques; Margarida Henriques; Teresa Fagulha; Fernanda Ramos; Tiago Luís; Miguel Fevereiro. Rabbit haemorrhagic disease virus 2 (RHDV2) outbreak in Azores: Disclosure of common genetic markers and phylogenetic segregation within the European strains. Infection, Genetics and Evolution 2015, 35, 163 -171.
AMA StyleMargarida Duarte, Carina Carvalho, Susana Bernardo, Silvia Barros, Sandra Benevides, Lídia Flor, Madalena Monteiro, Maria Isabel Marques, Margarida Henriques, Teresa Fagulha, Fernanda Ramos, Tiago Luís, Miguel Fevereiro. Rabbit haemorrhagic disease virus 2 (RHDV2) outbreak in Azores: Disclosure of common genetic markers and phylogenetic segregation within the European strains. Infection, Genetics and Evolution. 2015; 35 ():163-171.
Chicago/Turabian StyleMargarida Duarte; Carina Carvalho; Susana Bernardo; Silvia Barros; Sandra Benevides; Lídia Flor; Madalena Monteiro; Maria Isabel Marques; Margarida Henriques; Teresa Fagulha; Fernanda Ramos; Tiago Luís; Miguel Fevereiro. 2015. "Rabbit haemorrhagic disease virus 2 (RHDV2) outbreak in Azores: Disclosure of common genetic markers and phylogenetic segregation within the European strains." Infection, Genetics and Evolution 35, no. : 163-171.
A specific real time RT-PCR for the detection of RHDV2 was developed and validated using RHDV and RHDV2 RNA preparations from positive field samples. The system was designed to amplify a 127 nucleotide-long RNA region located within the vp60 gene, based on the alignment of six sequences originated in Portugal, obtained in our laboratory, and 11 sequences from France and Italy. The primers and probe target sequences are highly conserved in the vast majority of the RHDV2 sequences presently known. In the sequences showing variability, only one mismatch is found per strain, usually outlying the 3' end of the primer or probe hybridization sequences. The specificity of the method was demonstrated in vitro with a panel of common rabbit pathogens. Standardization was performed with RNA transcripts obtained from a recombinant plasmid harboring the target sequence. The method was able to detected nine RNA molecules with an efficiency of 99.4% and a R(2) value of 1. Repeatability and reproducibility of the method were very high, with coefficients of variation lower than 2.40%. The assay was proven a valuable tool to diagnose most of RDVH2 circulating strains, and may be also useful to monitor viral loads, and consequently, disease progression and vaccination efficacy.
Margarida Dias Duarte; Carina Carvalho; Silvia C. Barros; Ana M. Henriques; Fernanda Ramos; Teresa Fagulha; Tiago Luís; Elsa L. Duarte; Miguel Fevereiro. A real time Taqman RT-PCR for the detection of rabbit hemorrhagic disease virus 2 (RHDV2). Journal of Virological Methods 2015, 219, 90 -95.
AMA StyleMargarida Dias Duarte, Carina Carvalho, Silvia C. Barros, Ana M. Henriques, Fernanda Ramos, Teresa Fagulha, Tiago Luís, Elsa L. Duarte, Miguel Fevereiro. A real time Taqman RT-PCR for the detection of rabbit hemorrhagic disease virus 2 (RHDV2). Journal of Virological Methods. 2015; 219 ():90-95.
Chicago/Turabian StyleMargarida Dias Duarte; Carina Carvalho; Silvia C. Barros; Ana M. Henriques; Fernanda Ramos; Teresa Fagulha; Tiago Luís; Elsa L. Duarte; Miguel Fevereiro. 2015. "A real time Taqman RT-PCR for the detection of rabbit hemorrhagic disease virus 2 (RHDV2)." Journal of Virological Methods 219, no. : 90-95.
Margarida Duarte; Margarida Henriques; Silvia Barros; Teresa Fagulha; Fernanda Ramos; Tiago Luís; Miguel Fevereiro; Carina Carvalho; Sandra Benevides; Lídia Flor; Susana Bernardo. Detection of RHDV variant 2 in domestic rabbits in Azores. Veterinary Record 2015, 176, 499 -500.
AMA StyleMargarida Duarte, Margarida Henriques, Silvia Barros, Teresa Fagulha, Fernanda Ramos, Tiago Luís, Miguel Fevereiro, Carina Carvalho, Sandra Benevides, Lídia Flor, Susana Bernardo. Detection of RHDV variant 2 in domestic rabbits in Azores. Veterinary Record. 2015; 176 (19):499-500.
Chicago/Turabian StyleMargarida Duarte; Margarida Henriques; Silvia Barros; Teresa Fagulha; Fernanda Ramos; Tiago Luís; Miguel Fevereiro; Carina Carvalho; Sandra Benevides; Lídia Flor; Susana Bernardo. 2015. "Detection of RHDV variant 2 in domestic rabbits in Azores." Veterinary Record 176, no. 19: 499-500.