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- Crimean-Congo hemorrhagic fever virus circulating among sheep of Portugal: a nationwide serosurvey assessmentPublication . Mesquita, João; Cruz, Rita; Esteves, Fernando; Santos, Carla; Pousa, Humberto; Coelho, Catarina; Mega, Cristina; Nóbrega, Carmen; Vala, Helena; Peyrefitte, Christophe Nicolas; Nascimento, Maria São José; Barradas, Patrícia FerreiraCrimean-Congo hemorrhagic fever virus (CCHFV) is a widespread zoonotic pathogen that can cause mild to severe hemorrhagic disease in humans. CCHFV may be transmitted through direct contact with tissue or blood of viremic animals; however, the primary transmission route is through infected tick bites. CCHFV RNA has been detected in ticks feeding on domestic and wild animals in western Spain, suggesting an established circulation of CCHFV in Western Europe. Ruminants have been recognized as important CCHFV reservoirs and have been linked to human cases in endemic regions. Given the emergence of CCHF in neighboring Spain, and a report of two CCHFV seropositive humans in southern Portugal in 1985, we investigated the potential circulation of this virus in the country by performing a nationwide anti-CCHFV IgG serosurvey in sentinel sheep of Portugal. Sera (n = 459) randomly selected from widely distributed farms (n = 20) of Portugal were tested using a commercial double-antigen enzyme-linked immunosorbent assay, yielding an overall seroprevalence of 0.4% (95% confidence interval [CI] 0.04-1.56%). Positive sheep were from the southern region of Portugal (Alentejo region), which raise the seroprevalence of this region to 0.74% (95% CI 0.09-2.66%). This is the first study reporting the presence of CCHFV antibodies in sheep of Portugal, thus suggesting a geographical expansion of CCHFV to this country. It seems likely that CCHFV may exist focally in southern Portugal.
- Rickettsia massiliae circulation in sheep and attached Rhipicephalus sanguineus in Central PortugalPublication . Mesquita, João R.; Santos-Silva, Sérgio; de Sousa Moreira, Alícia; Baptista, Maria Beatriz; Cruz, Rita; Esteves, Fernando; Vala, Helena; Barradas, Patrícia F.Rickettsiosis is considered an emerging/re-emerging vector-borne disease that causes signifcant public health threats. Ticks are reservoirs and vectors of Rickettsia having a signifcant role in the transmission of rickettsiae. In Portugal, little is known about tick-borne Rickettsia species in sheep. Therefore, this study aimed to investigate rickettsiae infection in ticks and their sheep host from 27 farms in four districts of central Portugal, to clarify the role of the sheep host in the circulation of this zoonotic agent. Between March and May 2021, EDTA blood samples (n=100) of healthy grazing sheep and their ticks (n=100, one tick per animal) were collected during a herd health program in central Portugal. Obtained ticks were identifed as Rhipicephalus sanguineus sensu lato by PCR targeting a partial sequence of 16S rRNA gene followed by sequence analysis. Rhipicephalus sanguineus s.l. and host sheep blood were tested for the presence of Rickettsia spp. by PCR targeting a partial sequence of ompB and ompA genes. From a total of 100 paired R. sanguineus s.l. and host sheep, Rickettsia massiliae was detected in 62 ticks and 35 grazing sheep blood samples, collected in central Portugal, 2021. All 35 positive sheep had attached positive R. sanguineus s.l., with matching nucleotidic sequences. These fndings suggest that sheep may develop rickettsiemia and are likely capable of transmitting and amplifying the infection to uninfected ticks maintaining rickettsiae in circulation in the domestic cycle.
- SalivaPrint: sheep saliva electrophoretic protein profile in a bioinformatics approachPublication . Esteves, E.; Fernandes, M.; Cruz, I.; Esteves, Fernando; Rosa, N.; Correia, M. J.; Vala, Helena; Barros, M.Introduction: Sheep saliva is a fluid with properties relevant for the evaluation of individual wellbeing and disease states. It can be used for individual monitoring and decision aiding in therapeutic intervention. The total protein profile of each subject (SalivaPrint) can be integrated with clinical and environmental data to stratify individuals and analyze their health status. This strategy can also support the discovery of novel biomarkers. This study applies a bioinformatics strategy in order to identify standard protein profiles in healthy sheep. Standard total protein profiles of sheep are compared to human profile. Methods: Saliva samples were collected using an adaptation of the collection method available in Rosa et al 2016.Total protein profile of sheep saliva samples was performed b capillary electrophoretic analysis using the Experion™ technology. An algorithm which is able to identify common molecular weights ranges in a group of electrophoretic profiles was used. The molecular weight ranges identified were then used to discern between sheep and human population. Common molecular weights present in sheep SalivaPrint were then used with OvisOme database to identify proteins. Using AgBase GORetriever tool it was possible to analyze and catalogue their biological process. Results: Sheep and Human SalivaPrints are different and it is possible to discern both species. The SalivaPrints of individuals from the same flock appear to have a higher degree of similarity than individuals of different flocks. Sheep SalivaPrints showed five commonly present molecular weight ranges 7,9-8,9; 8,9-9,8; 12,7-13,6; 17,4 18,4 and 27,928,8 kDa. In this ranges, 14 proteins were identified using OvisOme database. The most representative biological process found was the response to stress (imm ne system process) with the proteins: Cathelicidin-1, Beta 2 microglobulin, Cathelin, Kallirein and Chloride intracellular channel protein. Conclusion: It was possible to conclude that different species have different salivary protein profiles. In addition to this, the total protein profile seem to be highly conserved between flocks. By using SalivaPrint profiles with bioinformatic tools like OvisOme.
- Serological Evidence for Schmallenberg Virus Infection in Sheep of Portugal, 2014Publication . Esteves, Fernando; Mesquita, João; Vala, Helena; Abreu-Silva, Joana; van der Poel, Wim H M; Nascimento, Maria S JBetween November and December of 2014, a serosurvey was set up to evaluate the presence of Schmallenberg virus (SBV) antibodies in sheep of Portugal. Sera (n = 1068) were tested using an indirect enzyme linked immunosorbent assay (ID Screen(®) Schmallenberg virus indirect, IDvet Innovative Diagnostics, Montpellier, France). The estimated occurrence of immunogobulin G (IgG) antibodies against SBV in sheep of Portugal was 12.8% (95% confidence interval 11.0-15.0%). This is the first study reporting the presence of SBV antibodies in sheep of Portugal.
- Molecular evidence of sporadic Coxiella burnetii excretion in sheep milk, central PortugalPublication . Pires, Humberto; Santos-Silva, Sérgio; Cruz, Andreia V.S.; Cardoso, Luís; Lopes, Ana Patrícia; Pereira, Maria; Nóbrega, Carmen; Mega, Cristina; Santos, Carla; Cruz, Rita; Esteves, Fernando; Vala, Helena; Matos, Ana Cristina; Barradas, Patrícia F.; Coelho, Ana Cláudia; Mesquita, João R.Coxiella burnetii is the etiologic agent of Q fever, a worldwide zoonosis. Cattle, sheep and goats are considered the main reservoirs of the disease. Transmission to humans occurs mainly through the inhalation of infectious aerosols from milk, faeces, urine, and birth products from infected ruminants. In this study, a 2-year longitudinal approach was performed to ascertain the excretion of C. burnetii in bulk tank milk samples of sheep from a mountain plateau in central Portugal, with sampling conducted during the years 2015 and 2016. From a total of 156 bulk tank milk samples tested by qPCR, only one showed to be positive for C. burnetii (1.28% [95%CI: 0.03–6.94]), from 2015, the first year of collection. Bidirectional sequencing and phylogenetic analysis of IS1111 transposase partial region confirmed the presence of C. burnetii DNA. The presence of C. burnetii in raw milk samples highlights the necessity for additional research to determine if raw milk is a potential source for human infection. Animal health surveillance and prevention measures against this zoonotic disease should be considered.