Bovine tuberculosis transmission between domestic and feral species in Portugal: first molecular evidences in Mycobacterium bovis isolates from a farm in Alentejo

No âmbito de um estudo mais alargado para a caracterização molecular de estirpes de Mycobacterium bovis (M. bovis) e de Mycobacterium caprae (M. caprae) isoladas em Portugal, tipificaram-se dois isolados de M. bovis provenientes de um bovino e de um javali (Sus scrofa), que coabitavam na mesma exploração na região do Alentejo. Duas metodologias, reconhecidas pela sua reprodutibilidade e poder discriminatório, foram utilizadas na genotipagem: o "spoligotyping" e a análise de MIRU-VNTR, caracterizando nove loci do genoma, que variam no número de repetições nucleotídicas contíguas: VNTR 3232, ETR-A, ETR-B, MIRU-26, QUB 11b, QUB 11a, ETR-C, VNTR 4156 e MIRU-4. Ambos os isolados apresentaram idêntico padrão spoligotyping SB0265, pouco frequente em Portugal mas já descrito em Espanha e em França, e igual número de repetições em cada um dos nove loci estudados. Estes dados, aliados à proximidade espacial dos animais, apontam para a probabilidade de transmissão de M. bovis entre eles, constituindo as primeiras evidências em Portugal de partilha de estirpes entre espécies silvestres e domésticas. Estes resultados deverão incentivar estudos mais aprofundados sobre o papel das espécies silvestres na epidemiologia da tuberculose bovina em Portugal

Tuberculose bovina: detecção molecular e genotipagem de Mycobacterium bovis

Nesta dissertação, pretendeu-se avaliar e aplicar um conjunto de ferramentas moleculares a estirpes de Mycobacterium bovis e Mycobacterium caprae, com o objectivo de melhorar o diagnóstico da tuberculose bovina e de proceder à primeira genotipagem destas estirpes em Portugal, clarificando vias de transmissão e a eventual existência de reservatórios silvestres. A extracção de DNA por disrupção mecânica/ lise enzimática e o PCR-REA gyrB produziram resultados que permitiram aumentar a sensibilidade e especificidade do diagnóstico, e identificar um conjunto aleatório de isolados (N=293), de várias regiões do país, com vista à sua genotipagem. O spoligotyping permitiu atestar da elevada diversidade dos padrões Portugueses (Hunter-Gaston Index; h=0,9) e compará-los com outros países. A análise dos polimorfismos dos MIRU-VNTR permitiu alcançar uma superior discriminação (h=0,98), especialmente nos “spoligotypes” mais frequentes, permitindo comprovar, em consonância com dados das explorações, a transmissão entre efectivos bovinos e entre bovinos e espécies cinegéticas da mesma área. Pelos seus índices discriminatórios elevados, as duas técnicas de genotipagem demonstraram a sua utilidade futura para estudos de epidemiologia molecular, integrando estratégias mais eficazes de erradicação em Portugal

O papel dos Larídeos como portadores e disseminadores de Salmonella spp.

A população de Larídeos tem aumentado grandemente devido a uma extrema adaptabilidade destas aves a novos habitas e fontes de alimento. As espécies mais frequentemente encontradas na nossa costa são Larus cachinnans, a gaivota-de-pata-amarela e Larus fuscus, a gaivota-de-asa-escura. Existem vários estudos que revelaram prevalências elevadas nos Larídeos. O desenvolvimento deste trabalho teve por objectivos contribuir para a avaliação do papel destas aves como portadores de Salmonella, visto no nosso país, existir uma notória carência de dados epidemiológicos, nomeadamente frequência de infecção, componentes ambientais e vias de transmissão. Num total de 306 amostras de fezes, 37 amostras foram positivas (12,1%). Os serotipos encontrados foram: S. Typhimurium (36,8%), S. Derby (18,4%); S. Enteritidis (10,5%); S. Agona e S. Hadar (7,9%); S. Goettingen, S. Newport e S. Virchow (5,3%); S. Bardo, S. Anatum, S. Infantis, S. Ohio, S. Orion e S. II l,4,12,27:b:- (2,6%). Os isolados com antibioresistências (68,7%), corresponderam a 24 perfis diferentes. Foram encontrados dois tipos fágicos diferentes de S. Typhimurium 5+: PT additional 10 (6 isolados) e PT 12 (5 isolados). Os resultados demonstram que as gaivotas podem ser portadoras e disseminadoras de Salmonella spp., transportando germes entre áreas adjacentes devido à sua mobilidade. Os serotipos, fagotipos e perfis de antibioresistências encontrados, mostram que as estirpes isoladas não são próprias dos Larídeos. Torna-se possível, devido a natureza detritívora destas aves, adoptá-las como modelos indicadores de contaminação ambiental, denunciando eventuais deficiências no confínamento e/ou tratamento de resíduos sólidos e efluentes de uma determinada área geográfica

O papel dos Larídeos como portadores e dissiminadores de Salmonella SPP

Dissertação de Mestrado em Ciências do Mar - Recursos Marinhos apresentada ao Instituto de Ciências Biomédicas de Abel Salazar da Universidade do Port

O papel dos Larídeos como portadores de Salmonella e Listeria spp

As in other countries, the population of gulls (Laridae) has been increased exponentially in Portugal. To evaluate the public health implications of this development, two hundred and eighty-five samples of gulls’ faeces were investigated for two bacteria species of zoonotic importance, Salmonella spp. and Listeria sp. Thirty-seven (13.0 %) samples were positive for Salmonella spp. Amongst these, teh most common serovars were Salmonella Typhimurium (37.8%) and Salmonella Derby (18.9%). Simultaneously presence of two different serovars was detected in six samples. Twenty four different antibioresistance profiles were detected in Salmonella sp. isolates (68.7%).Phage-types found for Salmonella Typhimurium 5+ were PT 12 and U302. Listeria spp. were present in twenty-eight (9.8%) samples, seventeen of which had Listeria monocytogenes (6.0%). Other species isolated were Listeria seeligeri (0.7%), L. innocua (5.3%), and L.welshimeri (0.7%). Seven samples were co-contaminated with two or more species. A combination of serotyping, phage-typing, cadmium and arsenic sensitivities were used to subtype 18 of the L. monocytogenes isolates recognising at least 10 different strains. Both bacterial genera were simultaneously isolated from twelve samples (4.2%). Several epidemiological explantion and the possible significance as markers waste management in the geographical area studied are discussed

“Biomarkers in a One Health perspective: current knowledge of their ability to merge human and animal health

Biomarkers are nowadays essential tools to be one step ahead for fighting disease, enabling an enhanced focus on disease prevention and on the probability of its occurrence. Research in a multidisciplinary approach has been an important step towards the repeated discovery of new biomarkers. Biomarkers are defined as biochemical measurable indicators of the presence of disease or as indicators for monitoring disease progression. Currently, biomarkers have been used in several domains such as oncology, neurology, cardiovascular, inflammatory and respiratory disease, and several endocrinopathies. Bridging biomarkers in a One Health perspective has been proven useful in almost all of these domains. In oncology, humans and animals are found to be subject to the same environmental and genetic predisposing factors: examples include the existence of mutations in BR-CA1 gene predisposing to breast cancer, both in human and dogs, with increased prevalence in certain dog breeds and human ethnic groups. Also, breast feeding frequency and duration has been related to a decreased risk of breast cancer in women and bitches. When it comes to infectious diseases, this parallelism is prone to be even more important, for as much as 75% of all emerging diseases are believed to be zoonotic. Examples of successful use of biomarkers have been found in several zoonotic diseases such as Ebola, dengue, leptospirosis or West Nile virus infections. Acute Phase Proteins (APPs) have been used for quite some time as biomarkers of inflammatory conditions. These have been used in human health but also in the veterinary field such as in mastitis evaluation and PRRS (porcine respiratory and reproductive syndrome) diagnosis. Advantages rely on the fact that these biomarkers can be much easier to assess than other conventional disease diagnostic approaches (example: measured in easy to collect saliva samples). Another domain in which biomarkers have been essential is food safety: the possibility to measure exposure to chemical contaminants or other biohazards present in the food chain, which are sometimes analytical challenges due to their low bioavailability in body fluids, is nowadays a major breakthrough. Finally, biomarkers are considered the key to provide more personalized therapies, with more efficient outcomes and fewer side effects. This approach is expected to be the correct path to follow also in veterinary medicine, in the near future

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

Overview of Cattle Diseases Listed Under Category C, D or E in the Animal Health Law for Which Control Programmes Are in Place Within Europe

The COST action “Standardising output-based surveillance to control non-regulated diseases of cattle in the European Union (SOUND control),” aims to harmonise the results of surveillance and control programmes (CPs) for non-EU regulated cattle diseases to facilitate safe trade and improve overall control of cattle infectious diseases. In this paper we aimed to provide an overview on the diversity of control for these diseases in Europe. A non-EU regulated cattle disease was defined as an infectious disease of cattle with no or limited control at EU level, which is not included in the European Union Animal health law Categories A or B under Commission Implementing Regulation (EU) 2020/2002. A CP was defined as surveillance and/or intervention strategies designed to lower the incidence, prevalence, mortality or prove freedom from a specific disease in a region or country. Passive surveillance, and active surveillance of breeding bulls under Council Directive 88/407/EEC were not considered as CPs. A questionnaire was designed to obtain country-specific information about CPs for each disease. Animal health experts from 33 European countries completed the questionnaire. Overall, there are 23 diseases for which a CP exists in one or more of the countries studied. The diseases for which CPs exist in the highest number of countries are enzootic bovine leukosis, bluetongue, infectious bovine rhinotracheitis, bovine viral diarrhoea and anthrax (CPs reported by between 16 and 31 countries). Every participating country has on average, 6 CPs (min–max: 1–13) in place. Most programmes are implemented at a national level (86%) and are applied to both dairy and non-dairy cattle (75%). Approximately one-third of the CPs are voluntary, and the funding structure is divided between government and private resources. Countries that have eradicated diseases like enzootic bovine leukosis, bluetongue, infectious bovine rhinotracheitis and bovine viral diarrhoea have implemented CPs for other diseases to further improve the health status of cattle in their country. The control of non-EU regulated cattle diseases is very heterogenous in Europe. Therefore, the standardising of the outputs of these programmes to enable comparison represents a challenge.Cost Action SOUND CONTRO

MIRU-VNTR typing adds discriminatory value to groups of Mycobacterium bovis and Mycobacterium caprae strains defined by spoligotyping

The value of Mycobacterial Interspersed Repetitive Units-Variable Number Tandem Repeats (MIRU-VNTR) as a genotyping technique for Mycobacterium bovis and Mycobacterium caprae, has been confirmed in different countries and epidemiological scenarios. However, a standardized panel of loci has not yet been adopted for these species, since allelic diversity of each locus differs among countries. To determine the most discriminatory loci, a panel of 181 M. bovis and M. caprae strains representing 12 spoligotypes was created. The panel included strains from the three predominant spoligotypes previously isolated in Portugal; strains from spoligotyping group SB0140, prevalent in the British Isles but also detected in Portugal; strains from spoligotypes common to cattle and wildlife species and strains from the M. caprae spoligotyping group SB0157. MIRU-VNTR analysis of these strains, targeting 8 selected loci, produced 87 different profiles (h = 0.99), being VNTR3232, QUB11a, ETR-B and ETR-A the most discriminatory loci (h = 0.96). A single M. bovis spoligotyping group could be differentiated – up to 44 MIRU-VNTR profiles. These results emphasize the high genotype diversity of Portuguese isolates compared with other countries. MIRU-VNTR typing was superior to spoligotyping for identifying multi-genotype infected herds and the combination of the two genotyping methods by a hierarchical approach confirmed the genetic relatedness of M. bovis isolates between cattle and wildlife