Browsing by Author "Duarte, Margarida D."
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- Blood collection from the external jugular vein of Oryctolagus cuniculus algirus sedated with midazolam: live sampling of a subspecies at riskPublication . Abade dos Santos, Fabio A.; Carvalho, Carina L.; Peleteiro, M. Conceição; Gabriel, Sofia Isabel; Patrício, Rui; Carvalho, João; Cunha, Mónica V.; Duarte, Margarida D.In the last decades, the European wild rabbit, particularly the Oryctolagus cuniculus algirus, a keystone species in the Iberian Peninsula ecosystems, declined severely, raising concerns from the wildlife authorities. The hunting calendar in Portugal limits sampling collection to a narrow window of few months annually. Nevertheless, governmental wildlife protection laws allow live rabbit sampling for population and sanitary evaluations. The aim of this study is to adjust blood collection protocols from the external jugular vein (EJV) described for domestic rabbits to the wild rabbit. Collection of peripheral blood is problematic in the wild rabbit given its small body size and the reduced calibre of vessels but mostly its nervous disposition and fragility. We describe in detail a procedure for EJV blood collection in 30 wild rabbits after sedation with midazolam. Emphasis is given to protocol adjustments for wild rabbit. Heart rate, respiratory rate and body temperature were assessed before sedation, after sedation but before collection and after blood collection. Sedation onset took on mean (SD) 8 ± 2 min. The technique allowed the collection at least 1 ml of blood, a satisfactory volume for routine laboratory testing. The differences observed in heart and respiratory rates before and after blood collection were not statistically sig-nificant, indicating that no cardiorespiratory interference occurred due to venepuncture. Recovery from sedation took on mean (SD) 17 ± 2 min. All animals were set free during the first hour after blood collection. This work aims to demonstrate that blood collection under sedation is a safe and feasible procedure in wild rabbits when practiced by experienced veteri-narians. At no time, whatsoever, was the physiological homeostasis at risk and no injuries were inflicted on the animals. To our knowledge this report constitutes the first guided description of blood collection from the EJV in sedated O. c. algirusand the first collection of physiological parameters measured under different conditions.
- Co‐infection by classic MYXV and ha‐MYXV in Iberian hare (Lepus granatensis) and European wild rabbit (Oryctolagus cuniculus algirus)Publication . Abade Dos Santos, F.A.; Dalton, Kevin P.; Carvalho, Carina L.; Casero, Maria; Alvarez, Angel L.; Parra, F.; Duarte, Margarida D.Myxomatosis is an emergent disease in the Iberian hare (Lepus granatensis). In this species, the disease is caused by a natural recombinant virus (ha-myxoma virus [MYXV]) identified for the first time in 2018 and has since been responsible for a large number of outbreaks in Spain and Portugal. The ha-MYXV, which harbours a 2.8 Kb insert-disrupting gene M009L, can also infect and cause disease in wild and domestic rabbits, despite being less frequently identified in rabbits. During the laboratory investigations of wild leporids found dead in Portugal carried out within the scope of a Nacional Surveillance Plan (Dispatch 4757/17, MAFDR), co-infection events by classic (MYXV) and naturally recombinant (ha-MYXV) strains were detected in both one Iberian hare and one European wild rabbit (Oryctolagus cuniculus algirus). These two cases were initially detected by a multiplex qPCR detection of MYXV and ha-MYXV and subsequently confirmed by conventional PCR and sequencing of the M009L gene, which contains an ha-MYXV-specific insertion. To our knowledge, this is the first documented report of co-infection by classic MYXV and ha-MYXV strains either in Iberian hare or in European wild rabbit. It is also the first report of infection of an Iberian hare by a classic MYXV strain. These findings highlight the continuous evolution of the MYXV and the frequent host range changes that justify the nonstop monitoring of the sanitary condition of wild Leporidae populations in the Iberian Peninsula.
- Harmless or Threatening? Interpreting the Results of Molecular Diagnosis in the Context of Virus-Host RelationshipsPublication . Abade dos Santos, Fábio A.; Portela, Sara J.; Nogueira, Teresa; Carvalho, Carina L.; de Sousa, Rita; Duarte, Margarida D.Molecular methods, established in the 1980s, expanded and delivered tools for the detection of vestigial quantities of nucleic acids in biological samples. Nucleotide sequencing of these molecules reveals the identity of the organism it belongs to. However, the implications of such detection are often misinterpreted as pathogenic, even in the absence of corroborating clinical evidence. This is particularly significant in the field of virology where the concepts of commensalism, and other benign or neutral relationships, are still very new. In this manuscript, we review some fundamental microbiological concepts including commensalism, mutualism, pathogenicity, and infection, giving special emphasis to their application in virology, in order to clarify the difference between detection and infection. We also propose a system for the correct attribution of terminology in this context.
- A potential atypical case of rabbit haemorrhagic disease in a dwarf rabbitPublication . Abade dos Santos, Fábio A.; Magro, Carolina; Carvalho, Carina L.; Ruivo, Pedro; Duarte, Margarida D.; Peleteiro, Maria C.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.
- A potential atypical case of rabbit haemorrhagic disease in a dwarf rabbitPublication . Abade Dos Santos, F.A.; Magro, Carolina; Carvalho, Carina L.; Ruivo, Pedro; Duarte, Margarida D.; Peleteiro, Maria C.ABSTRACT - 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.
- Recombinant myxoma virus infection associated with high mortality in rabbit farming (Oryctolagus cuniculus)Publication . Abade Dos Santos, F.A.; Carvalho, Carina L.; Monteiro, Madalena; Carvalho, Paulo; Mendonça, Paula; Peleteiro, Maria C.; Duarte, Margarida D.ABSTRACT - Myxomatosis is an emergent disease in the Iberian hare, having been considered a rabbit disease for decades. Genome sequencing of the strains obtained from Iberian hares with myxomatosis showed these to be distinct from the classical ones that circulated in rabbits since the virus introduction in Europe, in 1952. The main genomic difference in this natural recombinant hare myxoma virus (ha-MYXV) is the presence of an additional 2.8 kb region disrupting the M009L gene and adding a set of genes homologous to the myxoma virus (MYXV) genes M060R, M061R, M064R, M065R and M066R originated in Poxviruses. After the emergence of this recombinant virus (ha-MYXV) in hares, in the summer of 2019, the ha-MYXV was not detected in rabbit surveys, suggesting an apparent species segregation with the MYXV classic strains persistently circulating in rabbits. Recently, a group of six unvaccinated European rabbits (Oryctolagus cuniculus cuniculus) from a backyard rabbitry in South Portugal developed signs of myxomatosis (anorexia, dyspnoea, oedema of eyelids, head, ears, external genitals and anus, and skin myxomas in the base of the ears). Five of them died within 24–48 hr of symptom onset. Molecular analysis revealed that only the recombinant MYXV was present. This is the first documented report of a recombinant hare myxoma virus in farm rabbits associated with high mortality, which increases the concern for the future of both the Iberian hare and wild rabbits and questions the safety of the rabbit industry. This highlights the urgent need to evaluate the efficacy of available vaccines against this new MYXV.
- Simple method for establishing primary Leporidae skin fibroblast culturesPublication . Abade dos Santos, F.A.; Carvalho, C. L.; Almeida, Isabel; FAGULHA, TERESA; Ramos, Fernanda; Barros, Sílvia C.; Henriques, Margarida; Luís, Tiago; Duarte, Margarida D.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.
- West Nile virus transmission potential in PortugalPublication . Lourenço, José; Barros, Sílvia C.; Zé-Zé, Líbia; Damineli, Daniel S. C.; Giovanetti, Marta; Osório, Hugo; Amaro, Fátima; Henriques, Ana M.; Ramos, Fernanda; Luís, Tiago; Duarte, Margarida D.; Fagulha, Teresa; Alves, Maria João; Obolski, UriIt is unclear whether West Nile virus (WNV) circulates endemically in Portugal. Despite the country's adequate climate for transmission, Portugal has only reported four human WNV infections so far. We performed a review of WNV-related data (1966-2020), explored mosquito (2016-2019) and land type distributions (1992-2019), and used climate data (1981-2019) to estimate WNV transmission suitability in Portugal. Serological and molecular evidence of WNV circulation from animals and vectors was largely restricted to the south. Land type and climate-driven transmission suitability distributions, but not the distribution of WNV-capable vectors, were compatible with the North-South divide present in serological and molecular evidence of WNV circulation. Our study offers a comprehensive, data-informed perspective and review on the past epidemiology, surveillance and climate-driven transmission suitability of WNV in Portugal, highlighting the south as a subregion of importance. Given the recent WNV outbreaks across Europe, our results support a timely change towards local, active surveillance.