Detection and Management of Pregnancy Outcomes in Cattle

Pregnancy success is a key factor in order for any beef producer to have a profitable operation. Subsequently, the ability to detect compromised pregnancies is of upmost importance. Currently, pregnancy-associated glycoproteins [PAG], which are secreted by the ruminant placenta, remain as the only chemical-based, pregnancy-specific detection method available on a commercial scale for cattle pregnancy diagnosis. Furthermore, PAG cannot be used for diagnosis until day 28 of gestation. Small noncoding RNAs, microRNAs [miRNAs], have been successfully used as biomarkers for certain human diseases and disorders. It is possible that pregnancy-associated miRNAs located in bovine serum and uterine flush fluid can be detectable prior to PAG. Additionally, management efforts, such as evaluating reproductive tracts and estrus expression, can be performed prior to breeding to potentially identify and select females with optimal fertility. The aim of the two studies is to determine if pregnancy-related miRNAs can be identified in bovine serum and uterine flush fluid on day 18 of gestation, and to determine if reproductive tract size and position scores [SPS] and estrus are useful predictors of fertility in Bos indicus and Bos taurus cows. The first study was able to identify two novel extracellular vesicle-miRNAs as being more abundant in pregnant heifers compared with non-pregnant heifers on day 18 of gestation in both serum and uterine flush fluid. The second study identified that pregnancy rate, but not pregnancy loss, is interactively influenced by reproductive tract SPS and estrus activity

Actinomyces pyogenes in Embryonic Loss in Cattle

Actinomyces pyogenes is one of the bacteria commonly found in the bovine reproductive tract and it has been considered not to be a primary pathogen. The bacteria has also been isolated from foetuses and pus after abortion but its role as a primary or secondary pathogen in bovine abortion has remained an area of controversy and as yet little experimental work has been carried out to determine its role. In this thesis the potential role of A. pyogenes in bovine embryonic loss is examined

Cytological endometritis in dairy cattle : new insights into pathogenesis, diagnosis and treatment

Tese de Doutoramento em Ciências Veterinárias, na especialidade ClínicaABSTRACT - In postpartum dairy cows, subclinical endometritis (SCE) is characterized by persistent endometrial inflammation, with profound detrimental effects on subsequent fertility. Despite the known role of adipokines regulating metabolism and inflammation, the association of adipokine signaling with SCE is still poorly understood. Moreover, the effect of SCE on endometrial transcription was mainly determined from biopsy-derived whole tissue, thus masking effects on cell type-specific gene transcription. In addition, despite the recognized improvement in immune function following n-3PUFA supplementation, the therapeutic potential of these fatty acids for SCE control is still to be determined. Therefore, the main objectives of this work were to assess the relationship between adipokines and SCE (Chapter III), to elucidate the effects of progesterone and SCE in the transcription profiles of endometrial glandular, luminal and stromal cells (Chapters IV, V), and the effects of feeding rumen-protected n-3PUFA on endometrial homeostasis and fertility in postpartum dairy cows (Chapter VI). The results showed that adiponectin in plasma and uterine fluid in addition to chemerin uterine fluid have high discriminatory power for the diagnosis of SCE. This work additionally evidenced that progesterone and SCE affect endometrial transcriptomic profiles in a cell type-specific manner. This work also evidenced that the previous presence of immune cells is still impacting the transcriptome of endometrial cells at the end of the voluntary waiting period and that recovery or persistence of inflammation is associated with transcription patterns involved not only in immune function but also in tissue remodeling, cell adhesion, and uterine receptivity. Furthermore, despite having no apparent effect on the endometrial inflammatory status at the end of the voluntary waiting period, dietary supplementation with n-3PUFA decreased calving to conception interval. Overall, this thesis establishes adiponectin as a suitable biomarker of SCE, able to predict the risk of persistence of inflammation in postpartum dairy cows, provides new insights into the persistence and recovery of SCE, thus presenting putative alternative therapeutic strategies. Moreover, this work substantiates dietary n-3PUFA as valid nutraceuticals to improve reproductive parameters in postpartum dairy cows.RESUMO - Endometrite citológica em vacas leiteiras : novos desenvolvimentos em patogénese, diagnóstico e tratamento - Em vacas leiteiras pós-parto, a endometrite subclínica (ESC) é caracterizada por inflamação persistente do endométrio, com profundos efeitos prejudiciais na fertilidade subsequente. Apesar do conhecido papel das adipocinas na regulação do metabolismo e da inflamação, a regulação destas moléculas em vacas com ESC é ainda pouco compreendida. Além disso, o efeito da ESC na transcrição de células do endométrio tem sido explorado a partir de biópsias endometriais totais, mascarando assim os efeitos na transcrição específica de cada tipo celular. Além disso, apesar da reconhecida modulação da função imune após a suplementação com ácidos gordos polinsaturados (PUFA) ómega-3, o potencial terapêutico dos mesmos para o controlo da ESC ainda não foi determinado. Assim, os principais objetivos deste trabalho foram avaliar a relação entre adipocinas e a ESC (Capítulo III), elucidar os efeitos da progesterona e ESC nos perfis de transcrição de células endometriais do estroma, epiteliais glandulares e epiteliais luminais (Capítulos IV, V), e os efeitos da suplementação com PUFA ómega-3 com protecção ruminal na homeostase endometrial e fertilidade em vacas leiteiras pós-parto (Capítulo VI). Os resultados mostraram que a adiponectina no plasma e no lavado uterino, além da quemerina no líquido uterino, possuem alto poder discriminatório para o diagnóstico de ESC. Este trabalho evidenciou também que a progesterona e a ESC afetam os perfis de transcrição endometriais das diferentes populações celulares de uma forma específica, que a presença prévia de leucócitos ainda afeta o perfil de transcrição das células endometriais no final do período voluntário de espera e que a recuperação ou persistência da inflamação está associada a padrões de transcrição envolvidos não apenas na função imunológica, mas também na remodelação tecidular, adesão celular e receptividade uterina. Além disso, apesar de aparentemente não produzir efeito sobre o estado inflamatório endometrial no final do período voluntário de espera, a suplementação com PUFA ómega-3 diminuiu o intervalo parto-concepção. No geral, esta tese estabelece a adiponectina como um biomarcador adequado de ESC, capaz de prever o risco de persistência de inflamação em vacas leiteiras no pós-parto, fornece novas perspectivas sobre a persistência e recuperação da ESC, apresentando assim novas hipotéticas estratégias terapêuticas. Além disso, este trabalho substancia os PUFA ómega-3 como nutracêuticos válidos para melhorar os parâmetros reprodutivos em vacas leiteiras no pós-partoN/

Transferable Odor Differentiation Models for Infectious Disease Diagnostics

__Abstract__ In recent years the interest for the application of an electronic nose (eNose) in medical Diagnostics is increasing. There is a need for this since eNose Diagnostics is non-invasive, easy to run, fast and cheap. The eNoses, now on the market, however, turn out to be unsuitable for large-scale application. This is mainly due to insufficient reproducibility of measurement results. In this work an eNose is used which is cheap and suited for mass applications. The usage of advanced temperature control guarantees the reproducibility between eNoses. In practice, this means that a once developed analysis model for a specific disease easily can be transferred to any number of other eNoses. Application of mass-produced components keeps the cost low. In the research it is shown that temperature variation is the main cause of the significant differences in measurement characteristic between the metal oxide sensors on which the eNose is based. To illustrate the practical applicability pilot studies are described for sepsis (bacterial infection of the blood), tuberculosis (TB) and metritis (infection of the uterus in cows). In the sepsis and metritis studies the measurements were conducted in the headspace of the blood cultures and uterus mucus respectively. In the tuberculosis study the exhaled breath of patients analyzed. For the sepsis diagnostics 30 eNoses are used to identify 11 to identify clinical relevant pathogens in blood. The eNose can significantly speed up the diagnostic process: on average 78% of the pathogens were correctly identified within 6-8 hours after inoculation in contrast to the 24 hours typically needed with the current methods. The TB-study was conducted in Dhaka (Bangladesh) with 3 eNoses. It turned out to be possible to distinguish between healthy people and those with active TB infection [sensitivity 93.5%, specificity 85.3%] but also to identify an active TB infection in a group of TB suspects [sensitivity 76.5%, specificity 87.2%]. These results are significantly better than the much-used screening test based on microscopy. Currently there is no objective diagnosis for metritis. A vet performs the diagnosis based on a number of characteristics such as temperature and appearance of the sample. The eNose proved to be more reliable and objective than a control panel of veterinarians [sensitivity 100%, specificity 91.6%]

PRECISION DAIRY FARMING TECHNOLOGY SOLUTIONS FOR DETECTING DAIRY COW DISEASE TO IMPROVE DAIRY COW WELL-BEING

Dairy cow health is multifactorial and complex. High producing dairy cows have been described as metabolic athletes, but metabolic and infectious diseases around calving affect many cows. These diseases have drastic negative effects on dairy cow well-being, milk production, and dairy farm economics. Early disease detection could potentially improve disease management, treatment, and future prevention techniques. The first objective of this research was to evaluate the use of activity, lying behavior, reticulorumen temperature, and rumination time determined by precision dairy farming technologies to detect transition cow diseases including hypocalcemia, ketosis, and metritis. The second objective was to evaluate the ability of activity, body weight, feeding behavior, lying behavior, milking order, milk yield and components, reticulorumen temperature, and rumination time determined by precision dairy farming technologies to predict clinical mastitis cases. The last objective of this research was to evaluate the precision dairy farming technologies used in Objective 3 to predict subclinical cases

Mechanisms of fertility failure in high yielding dairy cows

Reproductive tract inflammatory diseases (RTID) of dairy cows are common worldwide and have been associated with a decrease in reproductive performance. The aims of this thesis were: (1) to quantify the effect of RTID on reproductive performance using data from 80 dairy herds across England and Wales, and from the University of Nottingham Dairy Centre; (2) to determine the effect of endometritis on the normality of post-partum milk progesterone profiles; (3) to investigate the association between endometritis and luteal vasculature and protein expression of steroidogenesis enzymes ex vivo; (4) to determine the expression of LPS-associated receptors including TLR4, CD14 and MD-2 in bovine CL using multiplex PCR; (5) to investigate the dose dependent effects of LPS on luteal endothelial cell vasculature in vitro and protein expression of steroidogenesis enzymes. Data analysis of 59118 cows from 80 dairy herds across England and Wales showed 12% prevalence of RTID from 2000-2007 (P<0.001). Cows with RTID had significantly longer intervals from calving to both first service and to conception by about 5 days (P<0.001) and 22 days (P<0.001), respectively. Moreover, cows with RTID had a lower conception rate to 1st service by 14% (P<0.001) and required more services per conception about 1 service more (P<0.001). In addition, they were 1.2 times more likely to be exited from the herd (P<0.001), and had higher 305d milk yield (P<0.01). Furthermore, this study analysed data from 708 cows at Nottingham dairy centre showed about 15% of prevalence of RTID form 2008-2014 (P<0.001). Similar effects were observed in the Nottingham dairy centre. However, only the day to 1st service was significant (P<0.001). The association between endometritis and milk progesterone profiles was investigated to establish the importance of endometritis on postpartum ovarian activity. Endometritis increased the incidence of atypical progesterone profile with prolonged luteal phase being the most affected. In the ex vivo study, there was a negative association between presence of endometritis with luteal vasculature, CL size, luteal progesterone content and quantification of steroidogenic enzyme expression. Endometritis caused significant inhibition in the degree of luteal vasculature, progesterone content and protein expression of steroidogenic enzymes by corpora lutea. LPS has been implicated in influencing ovarian function. Multiplex PCR showed that LPS receptors (TLR4, CD14, and MD-2) are expressed in bovine CL at all stages of CL development, indicating that CL could be a target for LPS action. A physiological cell culture system was consequently utilised to examine the effect of LPS on luteal endothelial cell network formation, progesterone (P4) production by luteal cells and steroidogenic enzyme expression in vitro. Treatment with VEGFA/FGF2 increased progesterone production by luteal cells (P<0.001). Moreover, progesterone production increased significantly from day 3-9 of culture. Under both basal condition and/or angiogenic stimulus (VEGFA/FGF-2), LPS (0.01, 0.1, 1 and 10 µg/ml) had no effect on P4 production by luteal cells on day 3, 5, and 9 of culture. LPS significantly inhibited luteal endothelial cells network formation. This was due to inhibition of endothelial cell proliferation and increasing endothelial cell apoptosis (P<0.001). In summary, the adverse impact that endometritis has on dairy cow fertility was identified by this work. This work offers a greater understanding of the effect of endometritis on dairy cow’s reproductive performance, early luteal development and function. In particular, this work provides evidence of a novel effect of endometritis on bovine luteal development and adequacy. It also offers further awareness into the role of LPS in terms of its negative effect on luteal endothelial cell angiogenesis and steroidogenesis

On the trail of Mycobacterium avium subsp. paratuberculosis and Mycobacterium bovis in mainland Portugal: a microbiological and molecular survey in wildlife

Tese de mestrado em Biologia Molecular e Genética, apresentada à Universidade de Lisboa, através da Faculdade de Ciências, 2015A paratuberculose e a tuberculose bovina são duas doenças infeciosas causadas por micobactérias patogénicas, com uma elevada importância ao nível da saúde e bem-estar animal e que provocam perdas económicas substanciais associadas à diminuição de produtividade, limitações ao comércio de animais e de produtos de origem animal e o abate sanitário de animais infetados, no caso da tuberculose. Acresce que, devido ao potencial zoonótico dos respetivos agentes etiológicos e apesar de estar ainda por demonstrar inequivocamente a associação do agente da paratuberculose à doença de Crohn em humanos, é necessário reforçar o conhecimento sobre a epidemiologia destas doenças, nomeadamente através de uma melhor caraterização dos agentes patogénicos, os seus hospedeiros, as vias de transmissão e a patogenia. Mycobacterium avium subsp. paratuberculosis (MAP) é o microorganismo responsável pela paratuberculose ou doença de Johne, uma infeção crónica do trato gastrointestinal que afeta ruminantes em todo o mundo. Os ruminantes parecem ser o hospedeiro preferencial de MAP, contudo esta micobactéria já foi isolada a partir de amostras biológicas de um elevado número de outras espécies animais, domésticas e selvagens. Mycobacterium bovis (M. bovis) é o agente etiológico da tuberculose bovina. À semelhança de MAP, também o isolamento de M. bovis já foi reportado num elevado número de hospedeiros, incluindo animais domésticos e selvagens. Na Península Ibérica, o veado e o javali estão identificados como reservatórios de M. bovis em determinadas áreas geográficas de elevada prevalência, tendo um importante papel no ciclo epidemiológico da doença. Neste trabalho, pretendeu-se contribuir para o estudo da epidemiologia da paratuberculose e da tuberculose animal em Portugal, através da vigilância molecular e microbiológica de populações silvestres rastreadas de forma oportunística. Realizou-se a pesquisa de MAP e M. bovis em amostras biológicas de carnívoros, veados e javalis, doadas para fins científicos por várias entidades, e provenientes de atropelamento acidental, ações cinegéticas recreativas ou de correção de densidade de predadores devidamente autorizadas em Portugal continental. Cento e trinta e quatro amostras de fezes e 149 amostras de baço, provenientes de 225 animais silvestres, foram rastreadas para a presença de MAP. Os espécimes testados pertenciam a sete espécies diferentes, nomeadamente, sacarrabos (n=149), raposa (n=40), geneta (n=5), fuinha (n=4), texugo (n=4), javali (n=2) e veado (n=21). As matrizes biológicas utilizadas foram processadas para realização de cultura em meio Herrold‟s com e sem micobactina J, e para extração de DNA. O DNA extraído foi testado para a presença da sequência de inserção IS900, específica de MAP, através de nested PCR em tempo real. A presença da sequência IS900 foi detetada em amostras provenientes de 19 dos animais testados (8,4%; 95%CI: 5,47-12,8%), evidenciando a sua exposição àquele agente. Os animais positivos incluem nove sacarrabos, quatro raposas, uma fuinha, uma geneta, um javali e três veados, originários de várias localizações geográficas. A percentagem de animais positivos das espécies sacarrabos e raposa foi de 6,0% (95%IC: 3,2-11%) e 10% (95%IC: 4,0-23%), respetivamente. A deteção de MAP em veados sugere a existência de uma fonte ambiental e transmissão indireta, possivelmente através da existência de água e/ou vegetação contaminada com MAP, sendo que a identificação do microorganismo em amostras de carnívoros e javalis também sugere a possibilidade de contaminação do ambiente ou das presas, nas localizações geográficas de onde eram oriundos os animais positivos. Tendo em consideração a informação disponível na literatura, este é o primeiro estudo onde é detetada a presença de DNA de MAP em genetas. A deteção de DNA de MAP em animais provenientes de norte a sul de Portugal, também representa uma nova informação, uma vez que todos os dados publicados até ao momento, se reportam a animais selvagens oriundos da região centro e nordeste do país. Registou-se a deteção do DNA de MAP em ambas as matrizes biológicas, tratando-se este do primeiro estudo em que é reportada a deteção de MAP em fezes de animais selvagens. A deteção do microrganismo nas fezes sugere a sua excreção, que na forma viável poderá promover a continuação do ciclo infecioso. A análise estatística dos resultados evidenciou uma associação significativa (p<0,05) entre os animais positivos para a presença de MAP e a sua localização geográfica, sendo Faro, Aveiro e Viana do Castelo, os distritos com maior risco. Por outro lado, para as variáveis espécie, género, classe etária e tipo de matriz biológica analisada, não foram obtidas diferenças estatisticamente significativas. A caracterização molecular das amostras MAP-positivas foi realizada através da análise de MIRU-VNTR. Apesar de ter sido realizada uma otimização do protocolo aplicado, não foi possível obter um perfil alélico completo para nenhuma das amostras em análise. Contudo, alguns alelos foram observados pela primeira vez, e alelos múltiplos foram detetados para o VNTR X3, 7, 10, 25 e 292, o que pode ser explicado pela presença de uma co-infeção e/ou ser devido a uma mutação nos referidos loci. Registou-se ainda pela primeira vez a ocorrência de quatro alelos para o VNTR X3. Relativamente à pesquisa de M. bovis, foram analisadas 23 amostras de fezes e 98 amostras de fígado, provenientes de 121 animais. Os animais utilizados pertenciam a cinco espécies diferentes, especificamente sacarrabos (n=93), raposa (n=4), fuinha (n=1), javali (n=2) e veado (n=21). A metodologia seguida foi semelhante à aplicada para a pesquisa de MAP, contudo neste caso as matrizes biológicas utilizadas foram processadas para realização de cultura em meio Lowenstein-Jensen com piruvato e Stonebrink, e o DNA extraído foi testado através de uma abordagem de PCR em tempo real semi-nested para a presença da sequência de inserção IS6110, a qual é específica para os membros do complexo Mycobacterium tuberculosis. M. bovis foi isolada a partir de uma amostra de fezes de um veado, proveniente do distrito de Castelo Branco, uma área geográfica pertencente à zona epidemiológica de risco definida pelas autoridades Portuguesas para a tuberculose em caça maior. Nenhuma das restantes amostras em estudo demostrou ser positiva por cultura ou PCR. O presente estudo vem também alargar o conhecimento sobre os genótipos de M. bovis e M. caprae que circulam em Portugal, e das relações epidemiológicas entre as diferentes espécies de hospedeiros, através da caraterização molecular de 127 isolados por spoligotyping. Os isolados utilizados (117 de M. bovis e 10 de M. caprae) encontravam-se depositados no biobanco do INIAV, e são provenientes de animais selvagens (veado e javali) e domésticos (bovinos, caprinos e suínos). A elevada diversidade de estirpes existente em Portugal é confirmada, tendo sido obtidos 27 perfis distintos e um bom poder de discriminação do método (D=0,91). O spoligotipo mais frequente entre os isolados de M. bovis foi SB1174 (23%), enquanto SB0157 foi o único perfil obtido para todos os isolados de M. caprae. Quatro novos spoligotipos (SB2354-SB2357) foram identificados neste trabalho e adicionados à base de dados internacional (http://www.Mbovis.org). Os spoligotipos SB1269, SB1265, SB1375, SB0948 e SB1060 foram obtidos pela primeira vez em Portugal. Oito dos spoligotipos identificados (SB1174, SB1264, SB0122, SB0265, SB1266, SB1195, SB0295 e SB1483) são comuns a javalis e veados, e à exceção do SB1483, os animais compartilham a mesma origem geográfica, sugerindo uma fonte comum de infeção nestas áreas. Apesar das limitações inerentes a uma amostragem oportunística, o cálculo dos índices de diversidade Shannon-Wiener, Simpson e Berger-Parker, que combinam Informação sobre a ruiqueza e abundância de espécies, e dos estimadores não paramétricos de riqueza de espécies, chao 1 e chao 2, evidenciaram que, apesar do esforço de amostragem poder ser melhorado, o painel de espécies e número de espécimes incluídos neste estudo são adequados aos objetivos delineados, possibilitando inferências epidemiológicas acerca dos agentes patogénicos rastreados, particularmente no caso de MAP. Concluindo, a paratuberculose e tuberculose bovina são doenças com elevado impacto ao nível da saúde animal e da economia. As micobactérias patogénicas responsáveis por estas doenças circulam em animais domésticos e selvagens em Portugal continental, reforçando assim a necessidade de uma maior vigilância e revisão das medidas de controlo, de modo a favorecer a erradicação das mesmas.Paratuberculosis and bovine tuberculosis (bTB) are important infectious diseases of cattle caused by pathogenic mycobacteria, whose control present a challenge to livestock producers and veterinary authorities. These diseases represent a major problem for animal health and cause substantial economic losses associated with decreased productivity, limitations on animal trade and transactions of animal products, and slaughtering of infected animals in the case of bTB. Moreover, due to the zoonotic potential of these mycobacteria, and although the association of the paratuberculosis agent and Crohn's disease in humans is still ambiguous, it is necessary to strengthen the knowledge of the epidemiology of bTB and paratuberculosis, particularly the genetic signatures of the pathogens, their hosts, their routes of transmission and pathogenesis. Mycobacterium avium subsp. paratuberculosis (MAP) is responsible for causing paratuberculosis or Johne's disease, a chronic infection of the gastrointestinal tract that affects ruminants worldwide. Ruminants appear to be the preferred or natural host, but MAP has already been isolated from other animal species, both domestic and wild. Mycobacterium bovis (M. bovis) is the causative agent of bovine tuberculosis. Besides cattle, M. bovis can infect other domestic and wild species. In Iberian Peninsula, red deer and wild boar are known to act as maintenance hosts for M. bovis in hotspot areas. In this work, the occurrence of MAP and M. bovis in wild carnivores, wild boar and red deer was assessed based on the molecular screening and microbiological culture of tissue samples opportunistically obtained from road-killed or hunted animals in mainland Portugal. MAP detection was performed in feces and spleen samples from 225 animals, belonging to seven different species, namely Egyptian mongoose (n=149), red fox (n=40), common genet (n=5), stone marten (n=4), European badger (n=4), red deer (n=21) and wild boar (n=2), collected between 2005 and 2015. Samples were tested by culture in Herrold‟s egg yolk solid medium, supplemented with and without mycobactin J, and by nested real-time PCR to amplify IS900 MAP-specific sequence. The occurrence of MAP-positive animals, detected exclusively by PCR, was 8,4% (95%CI: 5,47-12,8%), which included nine Egyptian mongoose, four red fox, one stone marten, one common genet, one wild boar and three red deer, from several geographic locations. The percentages of MAP-positive Egyptian mongoose and red fox were 6,0% (95%CI: 3,2-11%) and 10% (95%CI: 4,0-23%), respectively. The detection of MAP-positive red deer suggests an environmental source and indirect transmission, possibly indicating the presence of MAP-contamined water/pasture; while the identification of MAP-positive carnivores and wild boar also suggest contamination of the environment and/or ingestion of infected prey. To our knowledge, MAP circulation in genets is reported for the first time. The presence of MAP-positive animals in several geographic locations, from north to the south of Portugal, also represents new data, since all the information published so far on wildlife is limited to animals from the center and northeast regions of mainland Portugal. MAP DNA was detected in both biological matrices under study, being this survey the first report of MAP DNA in feces from wildlife. These results suggest contamination of the surrounding environment promoting the continuation of the infectious cycle. The statistical analysis to identify risk factors for MAP exposure showed a significant association (p<0,05) between MAP-positive animals and their geographical location, being Faro, Aveiro and Viana do Castelo, the districts with increased risk. Other variables like species, gender, age class and type of organic matrix, were also analyzed, but no statistically significant differences were obtained. Genotyping of MAP-positive samples was performed through MIRU-VNTR analysis, and, although several optimization approaches were attempted, it was not possible to obtain a complete allelic profile for any of the positive samples. Despite this fact, some alleles are reported for the first time, and multiple alleles were detected in VNTR X3, 7, 10, 25 and 292, which suggest co-infection with more than one MAP strain and/or mutation in these particular loci. Moreover, the occurrence of four alleles in VNTR X3 was registered for the first time. The exposure to M. bovis was tested in feces and liver samples of 121 animals from five different species, namely Egyptian mongoose (n=93), red fox (n=4), stone marten (n=1), wild boar (n=2), and red deer (n=21) collected in 2012, 2013 and 2015, by culture in Lowenstein-Jensen pyruvate and Stonebrink solid media and using a semi-nested real-time PCR approach targeting IS6110. M. bovis was isolated from one red deer from Castelo Branco, a geographic area enclosed in the epidemiological risk area for bovine tuberculosis in big game animals. None of the other samples under analysis was positive by culture or PCR. The molecular characterization of M. bovis and M. caprae isolates from the bio-bank of INIAV was performed, by spoligotyping, disclosing the genetic diversity of 117 M. bovis and 10 M. caprae strains from domestic (cattle, sheep and pig) and wild animals (wild boar and red deer) isolated between 2011 and 2015. Spoligotyping exhibited a good discriminatory power (D=0,91) for the strains, revealing 27 different patterns. SB1174 was the most common spoligotype, accounting for 23% of the M. bovis isolates; and SB0157 was the only pattern obtained for M. caprae. Four new spoligotypes (SB2354-SB2357) were identified and deposited in the international M. bovis database (http://www.Mbovis.org). The patterns SB1269, SB1265, SB1375, SB0948 and SB1060 were obtained for the first time in Portugal. Eight patterns (SB1174, SB1264, SB0122, SB0265, SB1266, SB1195, SB0295 and SB1483) were common to wild boar and red deer and, with the exception of SB1483, the animals share the same geographical location, suggesting a common source of infection in those areas. Despite the constraints related to our opportunistic sample, calculation of the diversity indices Shannon-Wiener, Simpson and Berger-Parker, combining information about species richness and abundance, and of the non-parametric estimators of species richness, chao 1 and chao 2, evidenced that, although sampling efforts could be reinforced, the panel of sampled species and number of specimens included was adequate for the purposes of this study, enabling inference of the epidemiological situation for the pathogens surveyed, particularly in the case of MAP.Results from this work thus confirm that MAP and M. bovis circulate in livestock and widely distributed wildlife species from specific geographic regions of mainland Portugal and suggest the possibility of environmental contamination, reinforcing the need for increased surveillance and adjustment of control measures in order to enable successful eradication of these relevant diseases

The dairy herd health and productivity service

Herd health and reproduction have been ranked as top priorities for improving the future productivity and profitability of dairy enterprises. There is a multitude of genetic, disease, nutritional, management and physiological issues that can dramatically alter health and reproduction. It is generally agreed that a multidisciplinary approach is necessary if improvement is to be achieved. Herd health schemes have evolved in the dairy industry throughout the world but the uptake of such schemes in the UK has been low. The veterinary input to many dairy farms is at best routine fertility visits confined to examination of the reproductive tract, and at worst occasional emergency calls. The Dairy Herd Health and Productivity Service (DHHPS) was established to provide the opportunity for veterinary surgeons to lead a multidisciplinary team which can monitor health, fertility and production and can plan remedial action, when necessary. Over a period of twenty years it has continued to identify infertility, mastitis and lameness as the main reasons for the involuntary disposal of dairy cows, although on some farms BSE has been a major factor. The trend in the last few years has been for an increase in culling for reasons of disease rather than for age or yield. The average disease rates have remained consistently high with over 100 treatments per 100 cows each year. Infertility, mastitis and lameness are the most common disease problems on DHHPS farms.Blood profiling and condition scoring have demonstrated that at least a third of cows tested were mobilising excessive fat during the transition from the dry period to early lactation. The DHHPS found 34.3% of 9,235 dry cows, sampled 0- 14 days before calving and 28 per cent of 20,502 cows between 5-40 days post calving, had raised BHB blood concentrations. 30.6 per cent of the same dry cows and 21.9 per cent of the early lactation animals had elevated NEFA results.Blood urea was measured to reflect the current protein intake and the protein /energy balance of the ration. 14 per cent of 9325 dry cows and 9.5 per cent of 20502 of early lactation cows had low urea N (<1.7 mmol/1). 25 per cent of the cows sampled between 5-40 days post partum had blood urea nitrogen concentrations above 3.3mmol/l.Low magnesium levels were detected throughout the year. 9.2 per cent of 9235 dry cows and 7 per cent of 19,738 early lactation cows between 5-40 days calved had blood magnesium concentration <0.8 mmol/1.GSHPx was used as an indicator of selenium status. 16.3 per cent of 1,206 heifers and 4.9 per cent of 6,998 cows had low GSHPx results Metabolic profiling can assist in identifying possible nutritional imbalances, but is likely to be most effective when it is carried out as part of a full, systematic investigation of the nutritional status of the herd.A survey of farmers and veterinary surgeons who have been involved with DHHPS indicated that they are receiving considerable benefits from the service. The service, by combining techniques such as computerised data recording and pre-planned blood analysis, provides regular information on disease occurrence, adequacy of nutrition and management in relation to productivity and economic performance. The main reason for its success was the team approach. The DHHPS has facilitated the solving of complex problems by encouraging multidisciplinary participation and affords the opportunity for the veterinary practitioner to become involved in giving positive advice on animal health